kinase superfamily: (catalytic) domam structure and classification

The eukaryotic protein kinase superfamily: idnase. S . . 1

(catalytic) domam structure and classificationSTEVEN K.. HANRS* AND TONY HUNTER2*Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA; and

Molecular Biology and Virology Laboratory, The Salk Institute, San Diego, California 92186, USA

The eukaryotic protein kinases comprise one of thelargest superfamilies of homologous proteins andgenes. Within this family, there are now hundreds ofdifferent members whose sequences are known. Al-though there is a rich diversity of structures, regulationmodes, and substrate specificities among the proteinkinases, there are also common structural features.These conserved structural motifs provide clear indica-tions as to how these enzymes manage to transfer thephosphate of a purine nucleotide triphosphate to thehydroxyl groups of their protein substrates. Theauthors of this review have carried out a monumentaltask of analyzing and collating the amino acid se-quences of all reported protein kinases and definingthe conserved structural features that characterize theportion of these proteins that is responsible for theircatalytic activity. Comparison of the sequences in thecatalytic fragment of the protein kinases has been usedto arrange these enzymes in evolutionary trees thatgroup subfamilies of closely related enzymes. It is com-forting that the structural relationships that emergefrom these trees result in groupings that also reflectrelated functions. The work presented in this reviewseems to be an excellent example of the type of analy-sis that will become indispensable in the coming years,as more and more sequence information become avail-able to biologists as a result of the genome projects.

PROTEIN KINASES 6

576 0892-6638/9510009-0576/$O1.50.© FASEB

ABSTRACT The eukaryotic protein kinases make up alarge superfamily of homologous proteins. They are re-lated by virtue of their kinase domains (also known ascatalytic domains), which consist of “‘250-300 amino acidresidues. The kinase domains that define this group ofenzymes contain 12 conserved subdomains that fold intoa common catalytic core structure, as revealed by the3-dimensional structures of several protein-serine ki-nases. There are two main subdivisions within the super-family: the protein-serine/threonine kinases and theprotein-tyrosine kinases. A classification scheme can befounded on a kinase domain phylogeny, which revealsfamilies of enzymes that have related substrate specifici-ties and modes of regulation.-Hanks, S. K., Hunter, T.The eukaryotic protein kinase superfamily: kinase (cata-lytic) domain structure and classification. FASEB J. 9,576-596 (1995)

Key Words: protein-tyrosine k inase protein-serine hi-

nose. protein phosphorylation AMP-dependent protein kinose

THE EUKARYOTIC PROTEIN KINASE SUPERFAMILY

One of the largest known protein superfamilies is madeup of protein kinases identified largely from eukaryotic

sources. (The term superfamily will be used here to dis-tinguish this broad collection of enzymes from smaller,more closely related subsets that have been commonlyreferred to as families). These enzymes use the y-phos-phate of ATP (or GTP) to generate phosphatemonoesters using protein alcohol groups (on Ser andThr) and/or protein phenolic groups (on Tyr) as phos-phate acceptors. The protein kinases are related by virtueof their homologous kinase domains (also known as cata-lytic domains), which consist of 250-300 amino acidresidues (reviewed in refs 1-3; and see below). During thepast 15 years, previously unrecognized members of theeukaryotic protein kinase superfamily have been uncov-ered at an exponentially increasing rate and currentlyappear in the literature almost weekly. This pace of dis-covery can be attributed to the past development of mo-lecular cloning and sequencing technologies and, morerecently, to the advent of the polymerase chain reaction(PCR),3 which facilitated the use of homology-based clon-ing strategies. Consequently, about 200 different superfa-mily members (products of distinct paralogous genes)had been recognized from mammalian sources alone!The prediction made several years ago (4) that the mam-malian genome contains about 1000 protein kinase genes(roughly 1% of all genes) would still appear to be withinreason, and may even be an underestimate (5).

In addition to mammals and other vertebrates, cu-karyotic protein kinase superfamily members have beenidentified and characterized from a wide range of otheranimal phyla as well as from plants, fungi, and protozo-ans. Hence, the protein kinase progenitor gene can betraced back to a time before the evolutionary separationof the major eukaryotic kingdoms. The identification ofeukaryotic-like protein kinase genes in prokaryotes (6, 7)raises the possibility that the protein kinase progenitorgene might have arisen before the divergence ofprokaryotes and eukaryotes (see below). Studies of thebudding and fission yeasts, Saccharomyces cerevi.ciae andSchizosaccharomyces pombe, have been particularly fruitfulin the recognition of new protein kinases. In these geneti-

‘This article is based on an introductory chapter in the Protein

Kinase Facisbook, edited by D. G. Hardie and S. K. Hanks, publish-ed in 1995 by Academic Press, London.

2To whom correspondence and reprint requests should beaddressed, at: Molecular Biology and Virology Laboratory, TheSalk Institute, 10010 N. Torrey Pines Rd., La Jolla, CA 92037,USA.

5Abbreviations: PCR, polymerase chain reaction; PKA-Ca,type a cAMP-dependent protein kinase catalytic subunit; Cdk2,cyclin-dependent kinase 2; Erk2, p42 MAP kinase; APE,

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EUKARYOTIC PROTEIN KINASE SUPERFAMILY 577

cally tractable organisms, the powerful approach of mu-tant isolation and cloning by complementation has netteddozens of protein kinase genes required for numerousaspects of cell function (8). In many cases, vertebratecounterparts have now been found for these genes, lead-ing to a growing awareness that protein phosphorylationpathways that regulate basic aspects of cell physiologyhave been maintained throughout the course of eu-karyotic evolution.

Even though the overwhelming majority of protein ki-nases identified from eukaryotic sources belong to thissuperfamily, a small but growing number of such enzymesdo not qualify as superfamily members. Most of these arerelated to the prokaryotic protein-histidine kinase family(see below), which forms the sensor components of two-component signal transduction systems (9). Included inthis category are a putative ethylene receptor encoded bythe flowering plant ETR1 gene (10), the product of thebudding yeast SLN1 gene (11, 12) thought to be involvedin relaying nutrient information to elements controllingcell growth and division, the mitochondrialbranched-chain a-ketoacid dehydrogenase kinase (13),and the mitochondrial pyruvate dehydrogenase kinase(14). In prokaryotes, protein-histidine kinases phosphory-late aspartates in their target proteins, but except for thetwo dehydrogenase kinases that phosphorylate serine, theacceptor specificities of most of the eukaryotic proteinkinases of this type are not known. In addition to theseprotein kinases, the Bcr protein encoded by the breakpointcluster region gene involved in the Philadelphia chromo-some translocation (15) and the A6 kinase isolated byexpression cloning using an anti-phosphotyrosine anti-body (16) have kinase domains unrelated to any knowneukaryotic or prokaryotic kinase. In addition, true pro-tein-histidine kinases are known in eukaryotes. One suchenzyme has been extensively characterized from buddingyeast but not yet molecularly cloned (17), and so it is notclear whether this enzyme will belong to the protein ki-nase superfamily or use a novel structural principle forphosphotransfer.

What about the prokaryotes? It has been known foryears that protein phosphorylation events play key regu-latory roles in numerous bacterial cell processes includingchemota,cis, bacteriophage infection, nutrient uptake,and gene transcription (reviewed in refs 18, 19). Thebacterial protein kinases have been divided into threegeneral classes (20): 1) protein-histidine kinases such asthose functioning in two-component sensory regulatorysystems (strictly speaking, these are protein-aspartyl ki-nases, because autophosphorylation on His is an interme-diary step in phosphotransfer to an aspartate in theresponse-regulator protein) (9); 2) phosphotransferasessuch as those of the phosphoenol pyruvate-dependentphosphotransferase system involved in sugar uptake (21);and 3) protein-serine kinases such as isocitrate dehydro-genase kinase/phosphatase (22). Amino acid sequenceshave been determined for members of each class, and allare unrelated to the eukaryotic protein kinase superfa-mily.

Recently, however, true homologs of the eukaryoticprotein kinases have been identified from two species ofbacteria, Yersinia pseudotuberculosis (7) and Myxococcus xan-thus (6, 23). Are these special cases, or the first examplesof many such genes in prokaryotes? The eukaryotic-likeprotein kinase YpkA from the pathogenic enterobacteriaY. pseudotuberculosis is encoded by a plasmid essential for

the virulence of this infectious organism. In addition toYpkA, at least two other proteins encoded by genes resid-ing on the virulence plasmid exhibit high similarity toeukaryotic proteins. Thus, it seems likely that the viru-lence plasmid genes were transduced from a eukaryotichost by horizontal transfer. The myxobacterium M xan-thus presents a different and perhaps more intriguingpicture. Application of the PCR homology-based cloningstrategy revealed that at least eight genes encoding mem-bers of the eukaryotic protein kinase superfamily are pre-sent in the genome of this species (23). The myxobacteriaare unusual prokaryotes in that they undergo a complexdevelopmental cycle upon nutrient depletion, much likethat of the eukaryotic slime mold Dictyostelium. Given thatprotein kinases are commonly involved in regulatinggrowth and differentiation of eukaryotic cells, it is attrac-tive to speculate that the eukaryotic-like protein kinasesin M. xanthus are specifically involved in regulating theirdevelopmental cycle. Indeed, one of these kinases, Pkn 1,was shown to be required for proper fruiting body forma-tion. The same could be true for the eukaryotic-like pro-tein kinase PknA from Anabena (24). In keeping with thisidea, neither the PCR approach applied to Escherichia coli(23) nor extensive sequencing of the E. coli genome (now30% complete) has yielded eukaryotic-like protein ki-nases. Hence, genes encoding members of the eukaryoticprotein kinase superfamily may be present only in bacte-ria that can undergo a developmental cycle. However,unpublished reports of eukaryotic-like protein kinases inStreptomyces coelicolor, and in three species of Methanococ-cus, suggest that such genes are more widely expressedamong prokaryotes, and potentially these genes representthe ancestors for the entire eukaryotic protein kinase su-perfamily.

THE HOMOLOGOUS KINASE DOMAINS

The kinase domains of eukaryotic protein kinases impartthe catalytic activity. Three separate roles can be ascribedto the kinase domains: 1) binding and orientation of theATP (or GTP) phosphate donor as a complex with diva-lent cation (usually Mg2 or Mn2’); 2) binding and orien-tation of the protein (or peptide) substrate; and 3)transfer of the y-phosphate from A1’P (or GTP) to theacceptor hydroxyl residue (Ser, Thr, or Tyr) of the pro-tein substrate.

Conserved features of primary structure

The total number of distinct kinase domain amino acidsequences available is now approaching 400 (Table 1).Included in this total are the vertebrate enzymes encodedby distinct paralogous genes, their presumed functionalhomologs from invertebrates and simpler organisms (en-coded by orthologous genes), and those identified fromlower organisms and plants for which vertebrate equiva-lents have not been found. Conserved features of kinasedomain primary structure have previously been identifiedthrough an inspection of multiple amino acid sequencealignments (1-3) . The large number of sequences nowavailable precludes showing an alignment containing allknown kinase domains. Thus, in Fig. 1 only 60 differentkinase domain sequences are aligned. These are drawn,however, from the widest possible sampling of the super-family and thus provide a good representation of the

A-C-G GroupAGC-I. Cyclic nucleotide-regulated protein kinase family

A. Cyclic AMP-dependent protein kinase (PEA) subfamilyvertebrate:

1. PEA-Ca:2. PKA-C:3. PEA-Or

Drosophila melanogaster1. DmPEA-CO:2. DmPEA-C1:3. DmPEA-C2:

Caenorhabditis elegans:1. CePKA:

Saccharomyces cerevisiae:

I. ScPKA-TpkI:Schizosaccharomyces pombe:

1. SpPKA1:Dictyostelium discoideum:

1. DdPEA:Aplysia calfornico:

1. ApIC: PEA catalytic subunit homolog2. Sak: “Spermatozoon-associated kinase”

B. Cyclic GMP-dependent protein kinase (PKG) subfamilyvertebrate:

1. PKG-I: PKG, type I* 2. PKG-II: PKG, type IIDrosophila melanogaster:

1. DmPKC-G1:2. DmPKG-G2:

PEA homolog

AGC-IL Diacyiglycerol-activated/phospholipid-dependent protein kinase C (PKC) familyA. “Conventional” (Ca” -dependent) protein kinase C (cPKC) subfamily

vertebrate:1. cPKCa:2. cPKCl:3. cPKC

Drosophila melanogaster:1. DmPKC-53Ebr: PKC homolog expressed in brain, locus 53E2. DmPKC-53Eey: PKC homolog expressed in eye, locus 53E

Aplysia cal[ornica:1. ApI-I: PKC homolog, type I

B. “Novel” (Ca ‘-independent) Protein Kinase C (nPKC) subfamilyvertebrate:

1. nPKC6:2. nPKCc:3. nPKCr:4. nPKCO:

Drosophila melanogaster:1. DmPKC-98F: PKC homolog, locus 98F

Aplysia cal!ft’rnica:1. ApI-H: PKC homolog, type II

Caenorhabditis elegans:1. CePKC: PKC homolog, product of tpa-1 gene

* 2. CePKCIB: PKC homolog expressed in neurons and interneuronsDictyostelium discoideum:* I. DdMHCK: PKC homologSaccharomyces cerevisiae:

1. ScPEAI:* 2. ScPEA2:Schizosaccharomyces pombe:

1. Pckl: “Pombe C-kinase”, type 12. Pck2: “Pombe C-kinase”, type 2

C. “Atypical” Protein Kinase C (aPKC) subfamilyvertebrate:

1. aPKC1:* 2.* 4.

‘More information about the individual protein kinases listed (including sequence references) can be obtained by contacting the authors or byconsulting The Protein Kinase Factsbook (42). Protein kinases marked with asterisks (*) were not included in the phylogenetic analysis due to theirrecent discovery. In many instances new protein kinases were cloned by more than one group; in these cases the most commonly accepted name isused for the entry and alternative names are listed in parentheses after the entry. Protein kinase homologs from DNA viruses are not included inthis classification.

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578 Vol. 9 May 1995 The FASEB Journal HANKS AND HUNTER

Table 1. Eukaiyotic protein kinase superfamily class[ication.

C. OthersDictyostelium discoideum:

1. DdPKI:

PEA catalytic subunit, alpha-formPEA catalytic subunit, beta-formPEA catalytic subunit, gamma-form

PEA catalytic subunit, CO formPEA catalytic subunit, Cl formPEA catalytic subunit, C2 form

PEA catalytic subunit homolog

PEA catalytic subunit homolog, type I

PEA catalytic subunit homolog

PEA catalytic subunit

PKG homolog, type IPKG homolog, type 2

Protein Kinase C, alpha-formProtein Kinase C, beta-formProtein Kinase C, gamma-form

Protein Kinase C, delta-formProtein Kinase C, epsilon-formProtein Kinase C, eta-formProtein Kinase C, theta-form

PKC homolog, product of PKCJ genePKC homolog, product of PKC2 gene

Protein Kinase C, zeta-formProtein Kinase C, iota-formProtein Kinase C, mu-form

Table 1. (continued).

D. Othersvertebrate:* 1.PKN:

I. RAC-ct:2. RAC-:

Drosophila:I. DmRAC:

caenorhabditis elegans:* I. CeRAC:

ACC-IV. Family of kinasese that phosphorylate C protein-coupled receptorsvertebrate:

I. ARK1:2. ARK2:3.RhK:

* 4.IT11:* 5.GRK5:

* 6. GRK6:Drosophila nwlanogaster.

I. DmGPRK1:2. DmGPRK2:

AGC-VI. Family of kinases that phosphorylate ribosomal S6 proteinvertebrate:

1. S6K: 70 kDa S6 kinase with single catalytic domain2. RSK1(Nt): 90 kDA S6 kinase, type 13. RSK2(Nt): 90 kDA S6 kinase, type 2

[Note: The RSK enzymes have two distinct catalytic domains. The Nt-domain is closely related to S6K, whereas theCt-domain is most closely related to phosphorylase kinase]

AC-VIII. Flowering plant “PVPKl Family” of protein kinase homologsPhylum Angiospmnophyta (Kingdom Plantae):

I. PvKI: Bean protein kinase homolog2. OsGl 1A: Rice protein kinase homolog3. ZmPPK: Maize protein kinase homolog4. AtPK5: Arabidopsis protein kinase homolog5. AtPK7: Arabidopsis protein kinase homolog6. AtPK64: Arabidopsis protein kinase homolog7. PsPKS: Pea protein kinase homolog

Other AGC-related kinasesvertebrate:

I. DMPK:2. Sgk:

* 3 Mast2O5:Neurospora crassa:

1. NcCot-1:Dietyostelium discoideum:

1. Ddk2:Saccharomyces cerevisiae:

1. ScSpkl:Phylum Angiospermophyta (Kingdom Plantae):* I. Atpkl: Arabidopsis protein kinase

CaMK GroupCaMK-I. Family of kinases regulated by Ca’7Calmodulin, and close relatives

A. Subfamily including “Multifunctional” Ca’/Calmodulin Kinases (CaMKs)vertebrate:

I. CaMK1:2. CaME2a:3. CaMK2fr4. CaMK2r5. CaMK2&:

* 6. EF2K:7. CaMK4:

Dual-specificity kinase


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AGC-III. Related to PEA and PKC (RAC) familyvertebrate:

AGC-V. Family of budding yeast AGC-related kinasesSaccharomyces cerevisiae:

I. Sch9:2. Ykr2:S. Ypkl:’

AGC-VII. Budding yeast Dbf2/2O FamilySaccharomyces cerevisiae:

1. Dbf2:2. Dbf2O:

Protein kinase with PKC-related catalytic domain

RAC, alpha-form; cellular homolog of v-Akt oncoproteinRAC, beta-form

RAC homolog

RAC homolog

-adrenergic receptor kinase, type 1(1-adrenergic receptor kinase, type 2Rhodopsin kinaseC-protein-coupled receptor kinase homologC-protein-coupled receptor kinase, type 5C-protein-coupled receptor kinase, type 6

Drosophila C-protein-coupled receptor kinase, type 1Drosophila C-protein-coupled receptor kinase, type 2

Suppressor of defects in cAMP effector pathwayAGC-related kinaseAGC-related kinase

Product of gene periodically expressed in cell cycleClose relative of DBF2 not under cell cycle control

“Myotonic Dystrophy Protein Kinase”“Serum and glucocortocoid regulated kinase”Spermatid “Microtubule-associated serine/threonine kinase”

Product of gene required for normal colonial growth

Product of developmentally-regulated gene

CaMK, type ICaMK, type II, alpha subunitCaMK, type II, beta subunitCaMK, type II, gamma subunitCaMK, type II, delta subunitElongation Factor-2 Kinase or CaMK type IIICaMK, type IV

Drosophila melanogaster:1. DmCaMK2:

Saccharomyces cerevisiae:1. ScCaMK2-1:2.ScCaMK2-2:

Aspergilins nidulans:I. AnCaMK2:

B. Subfamily including phosphorylase kinasesvertebrate:

I. PhK-yM: Skeletal muscle phosphorylase kinase catalytic subunit2. PhK-/T: Male germ cell phosphorylase kinase catalytic subunit3. RSK1(Ct): 90 kDa S6 kinase, type 1; C-terminal catalytic domain4. RSK2(Ct): 90 kDa S6 kinase, type 2; C-terminal catalytic domain

C. Subfamily including myosin light chain kinasesvertebrate:

1.skMLCK:2. smMLCK:3. Titin:

Caenorhabditis elegans:1. Twn:

Dictyostelium discoideum:1. DdMLCK: Slime mold myosin light chain kinase

D. Subfamily of plant kinases with intrinsic calmodulin-like domainPhylum Angiospermophyta (Kingdom Plantae):

1.CDPK: Soybean Ca2-regulatedkinase with intrinsic CaM-like domain2.AtAKI: Arabidopsis CDPK homolog

* 3 OsSpk: Rice CDPK homolog* 4. DcPk43l: Carrot CDPK homolog

E. Subfamily of plantkinaseswith highlyacidicdomain

Phylum Angiospermophyta (Kingdom Plantae):* 1.ASK1: Arabidopsis proteinkinase homolog with highly acidic idomain* 2. ASK2: Arabidopsis protein kinase homolog with highly acidic domain

F. Other CaMK-related kinasesvertebrate:

1.PskHl:* 2.Saccharomyces cerevisiae:

1.Mre4:* 2.Dunl:* 3.Rckl:* 4. Rck2:

CaMK-lI. Snfl/AMPK familyvertebrate:* 1:AMPK:

2: p78:Saccharomyces cerevisiae:

1.Snfl:2.KinI:3.Kin2:4. Yc124:

* 5.Ycl453:Schizosaccharomyces pombe:

1.SpKinl:2.NimI:

Phylum Angiospermophyta (Kingdom Planlae):1. PSnfl-RKIN 1: Rye putativeprotein kinase thatcomplements yeastsnfl polarity

2. PSnfl-AKIN1O: Arabidopsis putativeprotein kinase relatedto SNFI3. PSnfl-BKINI2: Barley protein related to SNFI

* 4. PKABA1: Wheat kinase induced by abscisic acid* 5 WPK4: Wheat kinase homolog regulated by light and nutrients* 6. NPK5: Tobacco Snfl homolog, activates SUC2 gene expression

Other CaMK Group KinasesPlasmodiumfalciparum (malarial parasite):

1. PfCPK:2. PfPK2:

C-M-G-C GroupCMGC-I. Family of cyclin-dependent kinases (CDKs) and other close relatives

vertebrate:Inducer of mitosis; functional homolog of yeast cdc2+/CDC28 kinases (Cdkl)Type 2 cyclin-dependent kinaseType 3 cyclin-dependentkinase

Type 4 cyclin-dependent kinaseType 5 cyclin-dependent kinase

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1. Cdc2:2. Cdk2:3.Cdk3:4.Cdk4:5. Cdk5:

CaMK-II homolog

CaMK-II homolog, product of CMKI geneCaMK-II homolog, product of CMK2 gene

CaMK-II homolog

Skeletal muscle MLCK (rabbit)Smooth muscle MLCK (rabbit)Huge protein implicated in skeletal muscle development

‘Twitchin” protein involved in muscle contraction or development

Putative protein-serine kinase“MAP Kinase-Activated Protein Kinase 2”

Protein required for meiotic recombinationProtein required for DNA damage-inducible gene expression‘Radiation sensitivity complementing kinase, type 1”“Radiation sensitivity complementing kinase, type 2”

“AMP-Activated Protein Kinase”Protein lost in carcinomas of human pancreas

Kinase essential for release from glucose repressionProtein kinase with N-terminal catalyticdomain

Close relative of KIN1Protein kinase homolog on chromosome IIIProtein kinase homolog on chromosome XI

Product of gene important for growth polarityInducer of mitosis

Ca’-regulated kinase with intrinsic CaM-like domainPutative protein kinase


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6. Cdk6: Type 6 cyclin.dependent kinase7. PCTAIRE1: Cdc2-related protein8. PCTAIRE2: Cdc2-related protein9. PCTAIRES: Cdc2-related protein10. Mo 15: “Cdk-activating kinase”; Negative regulator of meiosis (CAK)

Drosophila melanogaster:1. DmCdc2: Functional homolog of yeast cdc2+/CDC28 kinases2. DmCdc2c: Cdc2.cognate protein; Cdk2 homolog

Dictyostelium discoideum:I. DdCdc2: Functional homolog of yeast cdc2+/CDC28 kinases2. DdPRK: “Cdc2-related PCTAIRE Kinase”

A.spergillus nidulans:I. NIMXcdc2: Cdc2-related gene product

Plasmodiumfalciparum:1. PfPK5: Cdc2-related protein from human malarial parasite

Entamoeba histolytica:1. EhC2R: Cdc2-related protein

Crithidiafascicutata:I. CfCdc2R: Cdc2-related protein

L.eishmania mexicana:* I. LmCRK1: “Cdc2-Related Kinase”Saceharomyces cerevisiae:

1. Cdc28: “Cell-division-cycle” gene product2. Pho85: Negative regulator of the PHO system and cell cycle regulator3. Kin28: CDC28-related protein

Schizosaccharomyces pombe:I. SpCdc2: “Cell-division-cycle” gene product

Histoplasma capsulatum:* 1. HcCdc2: Cdc2 homolog from dimorphic fungusPhylum Angiosperntophyta (Kingdom Plantae):

I. Pcdc2: Flowering plant Cdc2 homolog othat complements yeast mutants* 2. MsCdc2B: Alfalfa Cdc2 cognate gene products that complements Cl/S transition

3. OsC2R: More distantly related Cdc2 homolog from riceCMGC-II. Erk(MAP kinase) family

vertebrate:1. Erkl: “Extracellular signal-regulated kinase”, type 1 (p44 MAP kinase)2. Erk2: “Extracellular signal-regulated kinase”, type 2 (p42 MAP kinase)3. ErkS: Somewhat distant relative of the Erk/MAP kinases

* 4. p63MAPK: Another more distant relative of the Erk/MAP kinases* 5 SAPK-a: “Stress-activated protein kinase, type alpha” (JNK2)* 6. SAPK-: “Stress-activated protein kinase, type beta”* 7. SAPK-/Jnk1: “Stress-activated protein kinase, type gamma” or “Jun N-terminal Kinase”* 8. p38: HOGI-related protein (MPK2)

Drosophila melanogaster:1. DmErkA: Homolog of Erk/MAP kinases; product of rolled gene

Caenorhabd it is elegans:* I. Sun: Erk/MAP kinaseSaccharomyces cerrevisiae:

I. Kss 1: Suppressor of sst2 mutant, overcomes growth al-rest2. FusS: Product of gene required for growth and matingS. S1t2: Product of gene complementing lyt2 mutants (MPK1)

* 4. Hogi: Product of gene required for osmoregulationSchizosaccharomyces pombe:

L Spkl: Product of gene that confers drug resistance to staurosporine, a PK inhibitorPhylum Deuteromycota (Kingdom Fungi):

1. CaErkl: Protein that interferes with mating factor-induced cell cycle arrestTrypanosoma btucei (Phylum Zoomastigina, Kingdom Protoctista):* KFR1: “KSSl- and FUSS-related” gene productPhylum Angiospermophyta (Kingdom Plantae):

1. PErk: Flowering plant Erk/MAP kinase homologs (7 distinct homologs identified in Arabidopsis)CMGC-IH. Glycogen synthase kinase 3 (GSK3) family

vertebrate:1. GSKSa: Glycogen synthase kinase 3, a-form2. GSK3: Clycogen synthase kinase 3, il-form

Drosophila melanogaster:1. Sgg: Product of shaggy/zeste-white 3 gene

Saccharomyces cerevisiae:1. Mckl: “Meiosis and centromere regulatory kinase”

* 2. ScGSK3 Protein closely related to MCK1* 3. MdsI: Dosage suppressor of mckl mutant

Dictyostelium discoideism:* 1. DdCSKS: Glycogen synthase kinase 3Phylum Angiosperniophyta (Kingdom Plantae):* 1. ASK-a: “Arabidopsis shaggy-related protein kinase”, type alpha* 2. ASK-i “Arabidopsis shaggy-related protein kinase”, type gamma

vertebrate:1. CK2a:1. CK2a’:

Drosophila melanogaster:1. DmCK2:

Caenorhabditis elegans:1. CeCK2:

Theileria parva (a protozoan parasite):1. TpCK2:

Dictyostelium discoideum:1. DdCK2:

Saccharomyces cerevisiae:1. ScCK2a:2. ScCK2a’:

Schizosaccharomyces pombe:* 1.SpCkal:Phylum Angiospmnophyta (Kingdom Plantae):

1. ZmCK2: Flowering plant casein kinase II, a-subunit homolog

Other CMGC Group kinasesvertebrate:

1. Mak:2. Ched:3. PITSLRE:4. KKIALRE:

* 5. PITALRE:* 6.

Saccharomyces cerevi.ciae:1. Sme 1: Product of gene essential for tart of niosis2. Sgvl: Kinase required for G-protein-mediated adaptive response to pheromoneS. Ctkl: Product of gene required for normal growth

Phylum Angiosperinophyta (Kingdom Plantae):* 1. Mhk: Arabidopsis thaliana “Mak homologous kinase”

Conventional Protein-Tyrosine Kinase Group (l-X: Non-membrane-spanning; XI-XXIH: Membrane-spanning)PTK-l. Src family

vertebrate:1. Src:2. Yes:3. Yrk:4. Fyn:5. Fgr:6. Lyn:7. Hck:8. Lck:9. BIk:

* 10. Frk:* 11.Rak:* 12. Fyk:

Drosophila melanogaster:1. DmSrc: Src homolog, polytene locus 64B

Dugeciai (Girardia) tigrina (Phylum Platyhelminthes):* 1. DtSpk-l: “Src-like planarianHydra vulgaris (Phylum Cnidaria):

1. Stk:Spongilla lacustris (Phylum Ponfera):

1. Srkl-4:

Src-related protein

Four distinct Src-related kinases

Protein-tyrosine kinase expressed in human breast tumors


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CMGC-IV. CIk familyvertebrate:

1. CIk:* 2.Srpkl:

S. PskGl:4. PskH2:

Drosophila melanogaster:* 1.Doa:Saccharomyces cerevisiae:

1. Yakl:2. Knsl:

Schiwsaccharomyces pombe:1. Dskl:

* 2. Prp4:

PTK-II. Brk familyvertebrate:* 1. Brk:

Casein kinase II, alpha subunitCasein kinase II, alpha-prime subunit

Casein kinase II homolog

Casein kinase II homolog

Casein kinase II a-subunit homolog

Casein kinase II, a-subunit

Casein kinase II, alpha subunit

Casein kinase II, alpha-prime subunit

Casein kinase II, a-subunit homolog (Orb5)

“Cdc-like kinase”Kinase that regulates intracellular localization of splicing factorsPutative protein kinasePutative protein kinase

Kinase encoded by “Darkener of Apricot” locus

Suppressor of RAS mutantNonessential protein kinase homolog

Disi-suppressing protein kinase implicated in mitotic controlPre-mRNA processing gene product; lacks subdomains X-X1

“Male germ cell-associated kinase”“Cholinesterase-related cell division controller”Galactosyltransferase-associated kinaseCdc2-nelated proteinCdc2-related kinaseCdc2-related kinase

Cellular homolog of Rous sarcoma virus oncoproteinCellular homolog of Yamaguchi 73 sarcoma virus oncoproteinYes-related kinaseProtein related to Fgr and YesCellular homolog of Gardner-Rasheed sarcoma virus oncoproteinProtein related to Fgr and YesHematopoietic cell protein-tyrosine kinaseLymphoid T-cell protein-tyrosine kinaseLymphoid B-cell protein-tyrosine kinaseFyn-related kinaseSTK-related kinase“Fyn and Yes-related kinase” from electric ray

1. Fak: “Focal adhesion kinase”

PTK-XI. Epidermal growth factor receptor familyvertebrate:

1. EGFR: Epidermal growth factor receptor2. ErbB2: Cell homolog of oncogene activated in ENU-induced rat neuroblastoma (Neu, HER2)3. ErbBS: Receptor tyrosine kinase related to EGFR (HERS)4. ErbB4: Receptor tyrosine kinase related to EGFR (Tyro2)

Drosophila melanogaster:1. DER: Homolog of EGF receptor

Caenorhabditis elegans:1. LET-2S: Product of gene required for normal vulval development

Schistosoma mansoni (Phylum Platyhelminthes):1. SER: EGF receptor homolog



SERIAL REVIEW

PTK-I1I. Tec familyvertebrate:

1. Tec:2. Emt:3. Btk:

* 4.Txk:Drosophila melanogaster:

1. DmTec:PTK-IV. Csk family

vertebrate:1. Csk:

* 2. MarK:PTK-V. Fes(Fps) family

vertebrate:1. Fes/Fps:2. Fer:

Drosophila melanogaster:1. DmFer:

PTK-VI. AbI familyvertebrate:

1. AbI:2. Arg

Drosophila melanogaster:1. DmAbl:

Caenorhabditis elegans:1. CeAbI:

PFK-VII. Syk/Zap7O familyvertebrate:

1. Syk:2. Zap7O:

Hydra vulgaris (Phylum Cnidaria):* 1. Htkl6:

PTK-VI1I.Jak familyvertebrate:

1. Tyk2:2.Jakl:S. Jak2:

* 4.JakS:Drosophila melanogaster:* l.Hop:

PTK-lX. Ackvertebrate:* 1. Ack:

PTK-X. Fakvertebrate:

PTK-XII. Eph/ElIc/Eck receptor familyvertebrate:

I. Eph:2. Eck:S. Eek:4. Hek:5. Sek:6. Elk:

* 7. Hek2:*

9. Cek5/Nuk:* 10.* 11.Ehk2:* 12.

‘Tyrosine kinase expressed in hepatocellular carcinoma”“Expressed mainly in T-cells” kinase (ltk, Tsk)“Bruton’s agammaglobulinaemia tyrosine kinase” (Emb)Tec-related protein.tyrosine kinase

Tec homolog, polytene locus 28C

“C terminal Src Kinase”; negative regulator of Src“Megakaryocyte-associated Tyr-kinase” (Hyl, Lsk, Ctk, Ntk)

Cellular homolog of feline and avian sarcoma viruses“Fes/Fps-related” kinase

Fer-related protein

Cellular homolog of Abelson murine leukemia virus“AbI-related gene” product

AbI-related protein

Nematode AbI-related protein

“Spleen tyrosine kinase”T-cell receptor “zeta chain-associated protein of 70 kDa”

Syk/Zap7O-related

Transducer of interferon c/I3 signals“Janus kinase”, type 1“Janus kinase”, type 2“Janus kinase”, type 3

Product of hopscotch gene required for establishing segmental body plan

“CDC42Hs-associated kinase”

Kinase detected in “erythropoeitin-producing hepatoma”“Epithelial cell linase”Eph/Fik-related protein-tyrosine kinaseEph/Elk related protein-tyrosine kinase (Cek4)“Segmentally-expressed kinase”“Eph-like kinase” detected in brain“Human embryo kinase” type 2 (CeklO)“Hepatoma transmembrane kinase”“Chicken embryo kinase 5”/”Neural kinase”“Eph homology kinase-l” (Cek7)“Eph homology kinase-2”“Mammary-derived tyrosine kinase, type 1”

“Anexelekto” (Cr. “uncontrolled”) tyrosine kinase (UFO, Ark)Cellular homolog of RPLSO avian oncoprotein (c-Ryk)“Brain tyrosine kinase”/”Sea related protein tyrosine kinase”/”Tyrosine kinase with Ig-likeand FN-III-like domains”/”Receptor sectaris” (TyroS)

PTK-XV. Platelet-derived growth factor receptor familyA. Subfamily witih 5 Ig-like extracellular domains

veitebrate:1. PDGFRa: Platelet-derived growth factor receptor, type alpha2. PDGFR: Platelet-derived growth factor receptor, type beta3. CSFIR Colony-stimulating factor-i receptor (c.Fms)4. Kit: Steel growth factor receptor5. Flk2: “Fetal liver kinase-2” (FltS)

B. Subfamily with 7 Ig-like extracellular domainsvertebrate:

1. FILl:2. F1t4:3. FIkI:

Cellular homolog of UR2 avian sarcoma virus oncoprotein

Product of sevenless gene required for R7 photoreceptor cell development


SERIAL REVIEW


* 13. Myk2:* 14. Cek9:* 15. Pag-* 16.Rtkl:* 17.* 18.

PTK-XIII. Axi familyvertebrate:

1. Aid:2. Eyk:

* 3

PTK-XIV. Tie/Tek familyvertebrate:

1. Tie:2. Tek:

PTK-XVI. Fibroblast growth factor receptor familyvertebrate:

1. FGFR1:2. FGFR2:S. FGFRS:4. FGFR4:

Drosophila melanogaster:1. DmFGFRI:

* 2.DmFGFR2:

P1’K-XVII. Insulin receptor familyvertebrate:

1. InsR:2. IGF1R:S. IRR:

Drosophila melanogaster:1. DmInsR:

PTK-XVIII. Ltk/Alk familyvertebrate:

1. Ltk:*

PTK-X1X. Ros/Sev familyvertebrate:

1. Ros:Drosophila melanogaster:

1. Sev:

PTK-XX. Trk/Ror familyvertebrate:

1. Trk:2. TrkB:S. TrkC:4. Ron:5. Ror2:6. TcRTK:

Drosophila melanogaster:* 1. Dror:

PTK-XXI. Ddr/Tkt family* 1.* 2. Tkt:

“Mammary.derived tyrosine kinase, type 2”“Chicken embryo kinase 9”“Pagliaccio” Xenopus protein expression in neural crest and neural tissuesZebrafish Eph/Elk-related protein-tyrosme kinaseZebrafish EpWElk-related protein-tyrosme kinaseZebrafish Eph/Elk-related protem-tyrosine kinase

‘Tyrosine kinase with Ig and EGF homology”‘Tumca interna endothelial cell kinase” (TIE2)

“Fms-liketyrosinekinase”,type I“Fms-like tyrosine kinase”, type 4“Fetal liver kinase-1” (KDR)

Fibroblast growth factor receptor, type 1 (FIg, Cekl)Fibroblast growth factor receptor, type 2 (Bek, K-SAM, CekS)Fibroblast growth factor receptor, type SFibroblast growth factor receptor, type 4

Fibroblast growth factor receptor homolog, type 1Fibroblast growth factor receptor homolog, type 2

Insulin receptorInsulin-like growth factor receptorInsulin receptor-related protein

Homolog of insulin receptor

“Leukocyte tyrosine kinase“Anaplastic lymphoma kinase

High molecular weight nerve growth factor receptorReceptor for nrain-derivedneurotrophic factorand neurotrophin-4/5Trk-related protein; receptor for neurotrophin-S“Ror” putative receptor, type I“Ror” putative receptor, type 2Trk-related receptor (electric ray)

Putative neurotrophic receptor

“Discoidin Domain Receptor” (TrkE, CAK, NEP, PtkS)“'Fyrosine Kinase Related to Trk” (Tyro 10)

SERIAL REVIEW


PTK-XXII. Hepatocyte growth factor receptor familyvertebrate:

1. HGFR: Hepatocyte growth factor receptor (MET)2. Sea: Cellular homolog of SIS avian erythroleukemia virus oncoproteinS. Ron: “Recepteur d’Origine Nantaise”

* 4. Stk: “Stem cell-derived tyrosine kinase”

PTK-XXIII. Nematode KinI5/l6 familyCaenorhabditis elegans:

1. CeKinl5: PTK expressed during hypodermal development2. CeKinl6: PTK expressed during hypodermal development

Other membrane-spanning protein-tyrosine kinases (each with no close relatives)vertebrate:

1. Ret: Normal homolog of oncoprotein activated by recombination2. Klg: “Kinase-like gene” product

* 5 Nyk/Ryk: “Novel tyrosine kinase-related protein” (VIK, Mrk, Nbtkl)Drosophila melanogaster

I. Torso: Product of torso gene required for embryonic anterior/posterior determination2. DmTrk: Distant relative of the mammalian trk gene

Marine sponge (Geodia cydonium):* 1. GCTK: Putative receptor PTK

Other protein kinase families (not falling into m ajor groups)0-I. Polo family

vertebrate:1. P1k: “Polo-like kinase”2. Snk: “Serum-inducible kinase”

* S. Sak: Polo-related kinase isolated in screen for genes regulating sialylationDrosophila melanogaster:

I. Polo: Protein kinase homolog required for mitosisSaccharomyces cerevisiae:

I. CdcS: Product of gene required for cell cycle progression

0-LI. MEE/STE7 familyvertebrate:

1. MEKI: “MAP ERK Kinase”, type I2. MEK2: “MAP ERK Kinase”, type 2

Drosophila melanogaster:1. Dsorl:

Saccharomyces cereoisiae:I. Ste7: Kinase required for haploid-specific gene expression2. Pbs2: Kinase required for antibiotic drug resistanceS. MkkI: “MAP Kinase Kinase”, type 1 (suppresses lysis defect of pkcl mutant)4. Mkk2: “MAP Kinase Kinase”, type 2 (suppresses lysis defect of pkcl mutant)

Schizo.saccharomyces pombe:1. Byrl: Kinase that suppresses rasl-mutant sporulation defect2. Wisi: Suppressor of cdc phenotype in triple mutant cdc2 5/wee i/win I strains

0-ILL. MEKK/SteI I familyvertebrate:* 1. MEKK: “MEK

Saccharomyces cerevisiae:1. Stell: Protein required for cell-type-specific transcription2. Bckl: “Bypass of C kinase” kinase

Schizosaccharomyces pombe:1. Byr2: Product of gene required for pheromone signal transduction

Phylum Angiospermophyta (Kingdom Plantae):* 1. NPK1: Flowering plant (tobacco) homolog of Bckl

O-IV. Pak/Ste2O familyvertebrate:* 1. Pak: “p21-(Cdc42/Rac) activated kinase”

Saccharomyces cerevisiae:1. Ste2O: Product of gene required for pheromone response

0-V. NimA familyvertebrate:

1. NekI: NimA-related kinase* 2. Nek2: NimA-related kinase* S. NetS: NimA-related kinase* 4. Nrk2: NimA-related kinase* 5. StkI: NimA-related

Aspergillus nidulans: -

1. NIMA: Cell cycle control protein kinaseDrosophila melanogaster:

1. Fused: Product of gene required for segment polarity


Gene product able to complement S. pombe wed mutant

Weel homolog from budding yeast

“Wee” size at division kinase; Cdc2 negative regulator“Mitosis inhibitory kinase”, negative regulator of Cdc2

“Heme-regulated eukaryotic initiation factor 2a kinase”“Double-stranded RNA-dependent kinase” (Tik)

Cellular homolog of retroviral oncogene productOncogenic protein closely related to c-RafOncogenic protein closely related to c-R.af

Type I receptor for activin and TGF-[I (Tsk7L, SKR1, ALK-2)Type I receptor for activin and TGFG-1 (ALK-1)Type I receptor TGF- (ALK-5)Type I receptor for activin (ALK-4)Type I receptor for BMP-2 and BMP-4 (ALK.3)“Activin receptor-like kinase”, type 6

Type I activin receptor homologProduct of saxophone gene

Type II receptor for activinType II receptor for activinType II receptor TGF-Putative receptor kinase expressed in gonads

Type II activin receptor homolog

Larva development regulatory protein; liMP receptor

“Mixed lineage kinase”, type 1“Mixed lineage kinase”, type 2“Mixed lineage kinase”, typeS (PTK1, SPRK)


SERIAL REVIEW


Trjpanosoma brucei (Phylum Zoomastigina, Kingdom Protoctista):I. NrkA: Trypanosome protein kinase related to NimA

Saccharomyces cerevisaie:I. KinS: Putative protein kinase

O-VJ. weel/miki familyvertebrate:

1. WeelHu:Saccharomyces cerevisiae:* I.Schizosaccharomyces pombe:

I.SpWeel:

2. MikI:

O-VII. Family of kinases involved in translational controlvertebrate:

1.HRI:2. PKR:

Saccharomyces cerevisiae:1. Gcn2: Protein required for translational derepression

0-VIlI. Raf familyvertebrate:

I.Raf-i:

2.A-Raf:3. B-Raf:

Drosophila melanogaster:I. DmRaf: Raf homolog

Caenorhabd it is elegans:1. CeRaf: Raf homolog; product of lin-45 gene required for vulval differentiation

Phylum Angiospermophyta (Kingdom Plantae):1. Ctrl: Negative regulator of ethylene response pathway

O-LX. Activin/TGF receptor familyA. Subfamily of type I receptors

vertebrate:1. ActR-l:

* 2. TSR-i:* 3.* 4.*

* 6.

Drosophila melanogaster:* I.*

B. Subfamily of type II receptorsvertebrate:

1.ActRII:

2.ActRIlB:3.TG9IRII:

*

Drosophila melanogoster:*

Caenorhabditis elegans:* i.DAF-4:

C. OthersCaenorhabditis elegana:

1. DAF- 1: Product of gene required for vulval development

O-X. Flowering plant putative receptor kinase familyPhylum Angiospermophyta (Kingdom Plantae):

1. ZmPK1: Putative receptor protein-serine kinase (maize)2. Srk: “S receptor kinase”; three distinct alleles: 2, 6, and 910 (Brassica)3. TmkI: Putative “Transmembrane receptor kinase” (Arabidopsis)4. Apki: Kinase that phosphorylates Tyr, Ser, and Thr (Arabidopsis)

* 5. Nak: “Novel Arabidopsis Kinase’ (Arabidopsis)6. Pro25: Putative kinase selected for specificity to thylakoid membrane protein (Arabidopsis)

* 7. Pto: Product of genen conferring pathogen resistance (tomato)* 8. Tmkl 1: Transmembrane protein with unusual kinase-like domain (Arabidopsis)* 9.PrkI: Pollen-expressed receptor-like putative kinase (Petunia)

0-Xl. Family of “mixed-lineage” kinases with leucine zipper domainvertebrate:* 1.MIki:* 2.M1k2:* 3.M1k3:

O-XiII. PKN family of prokaryotic protein kinasesMyxococcus xanthus (Phylum Myxobacteria: Kingdom Prohasyotae):

I. Pknl: Protein kinase homologous to eukaryotic kinases2. Pkn2: Protein kinase required for maintenance of stationary phase cells and development

Other protein kinase family members (each with no known close relatives)vertebrate:

I. Mos:2. Pimi:S. Cot:4. Esk:

* 5.GC* 6.Slk:* 7.LIMK:*

Drosophila melanogaster:

I. NinaC:2. Pelle:

* 3,Dictyostelium discoideum:

1. Sp1A:2. Dpyk2:

Ceratodon purpureus: (a moss)1. PhyCer:

Saccharomyces cerevisiae:1.Cdc7:2. CDCI5:3. Vpsl5:4. Nprl:5.Elmi:6.Irel:7. Yk15i6:

* 8.IpII:

Schizosaccharomyces pombe:1. Ran 1: Product of gene required for normal meiotic function2. Chki: “Checkpoint Kinase” that links rad pathway to Cdc2

* S. CskI: “Cyclin Suppressing Kinase”* 4. RPKI: “Regulatory cell proliferation kinase”

Entamoeba histolytica (Phylum Rhizopoda, Kingdom Protoctista):1. Ehmfkl: Distant relative of Mos

Phylum Angiospernsophyta (Kingdom Plantae):1. GmPK6: Protein kinase homolog (soybean)

* 2. Tsl: Product of Tousled gene required for normal leaf/flower development (Arabidopsis)Yersinia psuedotubereulosis (Phylum Omnibacteria, Kingdom Prokaiyotae):

1. YpkA: Enterobacterial protein kinase essential for virulence

SERIAL REVIEW



0-XII. Casein kinase I familyvertebrate:

I. CK1a:2. CKi:3. CK14. CKI8:

Saccharomyces cerevisiae:1. Ycki:2. Yck2:S. Hrr25:

Schizosaccharosnyces pombe:* I. Hhpi:* 2. Hhp2:

Casein kinase I, type alphaCasein kinase I, type betaCasein kinase I, type gammaCasein kinase I, type delta

Budding yeast casein kinase I homolog, type IBudding yeast casein kinase I homolog, type 2Kinase required for DNA repair

Fission yeast casein kinase I homolog, type 1Fission yeast casein kinase I homolog, type 2

Cellular homolog of retroviral oncogene productProto-oncogene activatedby murine leukemia virusProduct of oncogene expressed in human thyroid carcinoma“Embryonal carcinoma STY kinase”; dual specificity (PIT)Kinase expressed in germinal center B cellsSTE2O-related kinase“UM motif-containing kinase”‘Testis-specific kinase”

Product of gene essential for photoreceptor functionProduct of gene required for dorsalventral polarityProduct of gene required for rotation of photoreceptor clusters

Spore lysis A protein kinaseDevelopmentally-reguated tyrosine kinase, type 2

Putative protein-tyrosine kinase encoded by a phytochrome gene

“Cell-division-cycle” control gene product“Cell-division-cycle” control gene productProduct of gene essential for sorting to lysosome-like vacuoleProduct of gene required for activity of ammonia-sensitive amino acid permeasesProduct of gene required for yeast-like cell morphologyRequired for Myo-inositol synthesis and signaling from ER to the nucleusPutative protein kinase gene on chromosome XIProduct of gene required for chromosome segregation

known primary structures. The kinase domains are fur-ther divided into 12 smaller subdomains (indicated byRoman numerals), defined as regions never interruptedby large amino acid insertions and containing charac-teristic patterns of conserved residues (consensus line inFig. 1).

Twelve kinase domain residues are recognized as beinginvariant or nearly invariant throughout the superfamily(conserved in over 95% of 370 sequences), and hencestrongly implicated as playing essential roles in enzyme

function. Using the type a cAMP-dependent protein ki-nase catalytic subunit (PKA-Ca) as a reference point,these are equivalent to G1y50 and G1y52 in subdomain I,Lys72 in subdomain II, G1u91 in subdomain III, Aspl66and Asnl7l in subdomain VIB, Asp184 and G1y186 insubdomain VII, G1u208 in subdomain VIII, Asp22O andG1y225 in subdomain IX, and Arg280 in subdomain XI.

The patterns of amino acid residues found within sub-domains VIB, VIII, and IX have been particularlywell-conserved among the individual members of the dlif-

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Figure 1. Multiple alignments of 60 kinase domains representative of members of the eukaryotic protein kinase superfamily. Theabbreviated names used are as defined in Table 1. The single letter amino acid code is used and gaps are indicated by dashes. Theentire sequences for the larger inserts are not shown, but excluded residues are indicated as numbers in brackets. Twelve distinctsubdomams are indicated by Roman numerals. The consensus line is given according to the following code: uppercase letters, invariantresidues, lowercase residues nearly invariant residues; o, positions conserving nonpolar residues; *, positions conserving polarresidues; +, positions conserving small residues with near neutral polarity. Residues corresponding to the numbered n-strands (b)and a-helices (a) in PKA-Ca are indicated in the 2- structure line.

ferent protein kinase families and these motifs have beentargeted most frequently in PCR-based homology clon-ing strategies aimed at identifying new family members.

Relationship between conserved subdomains, higher

order structure, and catalytic mechanismThe homologous nature of the kinase domains impliesthat they all fold into topologically similar 3-dimensionalcore structures and impart phosphotransfer according toa common mechanism. The larger inserts found withinsome kinase domains are likely to represent surface ele-ments that do not disrupt the basic core structure. Withthe solution of the crystal structure of mouse PKA-Ca, ina binary complex with a pseudosubstrate peptide inhibi-tor (PKI 5-24; TTYADFIASGRTGRRNAIHD, the under-lined Ala substituting for the Ser phosphoacceptor), thegeneral topology of a protein kinase catalytic core sti-uc-

ture was revealed for the first time (25, 26). Later, struc-tures of ternary complexes of PKA-Ca, thepseudosubstrate inhibitor, and either MgATP orMnAMP-PNP (an MgATP analog) were solved (27, 28).As a consequence of these studies, precise functionalroles for most of the highly conserved kinase domainresidues have now been assigned.

The kinase domain of PKA-Ca folds into a two-lobedstructure (Fig. 2). The smaller, NH2- terminal lobe, whichincludes subdomains I-IV, is primarily involved in an-choring and orienting the nucleotide. This lobe has apredominantly antiparallel f-sheet structure that isunique among nucleotide binding proteins. The largerCOOH-terminal lobe, which includes subdomainsVIA-XI, is largely responsible for binding the peptidesubstrate and initiating phosphotransfer. It is predomi-nantly a-helical in content. Subdomain V residues span

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the two lobes. The deep cleft between the two lobes isrecognized as the site of catalysis. The crystal structuresof four additional eukaryotic protein kinase superfamilymembers-cyclin-dependent kinase 2 (Cdk2) (29), p42MAP kinase (Erk2) (30), twitchin kinase (31), and caseinkinase I (32)-have been reported more recently, and asexpected, their kinase domains were found to fold intotwo-lobed structures topologically very similar to thecatalytic core of PKA-Cct. Notable differences, however,were found in the regions corresponding to subdomainVIII in the Cdk2 and Erk2 structures, apparently reflect-ing the fact that these are structures of enzymes in aninactive state (see below). The twitchin structure is also ofan inactive enzyme, but in this case it is inactive due tothe presence of an autoinhibitory peptide sequence,which lies on the COOH-terminal side of the kinase do-main and folds back into the active site cleft between thetwo lobes (31). This peptide apparently forces the two

lobes to rotate almost 30#{176}with respect to one another,and in this configuration inactive twitchin is more similarto the open configuration of PKA-Ca without PKI (33).In both twitchin and Cdk2 the a-helix C in subdomainIII also adopts a different position to that of helix C inPKA-Ca. Unfortunately, no structure of a protein-tyro-sine kinase catalytic domain was available at the time ofwriting (see “Note added in proof”), but the ease withwhich it has been possible to model the kinase domain ofthe EGF receptor protein-tyrosine kinase on to that ofthe PKA-Ca emphasizes that the structure of the pro-tein-tyrosine kinases will be similar to that of the pro-tein-serine kinases (34)

The conserved kinase subdomains correspond quitewell to precise units of higher order structure. The func-tions of the individual subdomains will be discussedbriefly later on a subdomain.-by-subdomain basis, mak-ing reference to the crystal structure of PKA-Ca and

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O----KN9OKSF-EVDIWSWVWLTAI.LI-OEFPFQR RDv),FIYNEIXcRDF--SFPRCRP ISDROEILIRDILSLDPXREPSLTEUID YVOff-----0CXYYST-AVDIWSIOCIFA6’1E-RBALFPGDSEI---DQLFRIFRTWTPDEE-VWROVTlPDY5P5TP XNERQDTSRVVPP LRSLLs6LNYDEWIRIS51AM.A NPFT

NSxGYTX-SIDIWS50CI1AI,S-NRPIFRORHYL---DQUu6XL0ILGSPSQE-DU8-CIDIL59A18TLLSLPN95I5VPWLFW ACuI(AWLL(NLT?NPH5RIEVEQALA 0901.-----OA?VYTS-SID’,’WSAOCVLAELLL-OQFIFRODSGV---DQLVEIIRVLOTPTRE-QIR-DIIPWYItTR?PQIXAH---WIEVFESR TPP95.IALCSSLLETEPSSRLSPL,EACA HEFT

VSP!AWFLAY6Q8RLT51EANE 09SF169FF

5(24 )T55PLTR-5IDIFS)UCVTYYILS5095lPFY SREuIIIR0IFSLDCL16S LIAEATDLIRII8DPL5RPMjVLR HP1ZKCQAQST-KIDIWS%VILLSLLO-RRFPW?QSL }6YWCTQVL!QCFIDPSSAEDLLK 59FFWR6ST-5ADITS1A?LIlOQr-0TPPWVE RYPRSATPSYLYIIHKQAPPLEDIAED CSPlR.ISLNP0UIRPRAADL.L5 HR.L

-----lZASE-XSDPFLWSTLLHCIZ-GTm6PEI5P----NQQIFITSEPAJIVl95TPINRP0I?a VLTATIEFITDIIOVSAIWRPDSNEARL HEFt.Q0FtffSV-QSDWLSt.VlAV-ORYPIPPP(36)RPPNEIFnDTIVNEFPP95.PSGV TSLE DOT lCLROlPAA.5QUlV HASYQlVYSI-56lISLaIMIIELVr-OEFPIIaI DTPmfl..LQRIPSPRLPI TtE8FTDFVNECCmIERERSSIKELLN HDLI5QTAirA-KADIWS1ucVI69’T-6PFPDF SQNQAIFRIOIIITTPERPSW ATSlFL55AF01.DYQYRPSAI.ELLQ 0915.DffPYISI-ESDINEWCVLY01.CT-LEHAFER -G5qLV1XI90SFPPVSPH TSYDt.RSLLSQLT)IPRPSVNSILE ROT!AATL-RSOINE”CINYELCQ-REFPFNE RT0IQLVIRED5FAPLPOT TREE IVIASCL PIRPD?ATLIW SPY!AD!PTI-HAalSIaCIAYESW.OQPPFCA SSILHLVThIKHEDVUIP95 LTCECRSFLDO1.LDPosITQZ.LC HPFV

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-----ROT-LSDIWSLGATWD6LT-IPPYIII L1NITTAV016DTTYPPSS F9EPL50FLCTVIOOITERP17DQLL5 NW!FAxYDPR-PVDIWSSAIIFAC1tIL-PWRIP5l---rR1SFKLFCSORDCDSLSSLVrRTP0PPSYDESflSTRE5XPESS5lVSDP1*lVNIQPQ(5 )LPEETQHIIULLAPACR6IEEII6E PFWIYHRYH0R-SAWWSWXLLYlYC-ODIPF D1DERIIROQVTFVQR VSSQHLZ11L8LRPS05P’rFEEIQN HP8

5(24 )SSSERTA-PSIWWAIOIILDILCC-RPNPWR RACSQTDOTYRSSVNNPSTLLSILP ISRSU6SLUmIFNPRTRIThPELS? LVu8DI( 6 }CRSKISP-XSDYWS6OCILYYWTY-OXTPFQQXI NQISKLHAIIDRNHEIEFPDIP EKDDLQOVLECCLICRDPKQRISIPEU.A 169WW(4)DFVTF-RLDIWSLaVTLTCLLY-NELPFPGuIff---ETYHKIIEVSL.SSRIN ITu6r*.VImLssDVTLRIsIQoLVR VLSR-----PYH-LSD’11WVILYSLFE-LPFDPPPNE(7 )ATSNRIMIUIuIYRLSD YRT?IV05QIV8TLTRfl6QRWSINEIYE SPFV

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Figure 1 (contd.).

drawing attention to the proposed roles of the nearlyinvariant amino acid residues (25-27, 28) and other resi-dues of interest. For more detailed information, thereader is referred to recent reviews on the structure ofPKA-Ca (35-37) and to an excellent comparative reviewof the structures of PKA-Ca, Erk2, and Cdk2 (38).

Subdomain I, at the NH2 terminus of the kinase do-main, contains the consensus motif Gly-x-GIy-x-x-Gly-x-Val (starting with G1y50 in PKA-Ca). The kinase do-main NH2-terminal boundary occurs seven positions up-stream of the first glycine in the consensus, where ahydrophobic residue is usually found. Subdomain I resi-dues fold into a 13-strand-turn-f3-strand structure encom-passing 13-strands 1 and 2, and this structure acts as aflexible flap or clamp that covers and anchors the non-transferable phosphates of ATP. The backbone amides ofSer53, Phe54, and Gly55 form hydrogen bonds with ATP13-phosphate oxygens. Leu49 and Val57 contribute to ahydrophobic pocket that encloses the adenine ring ofATP.

Subdomain II contains the invariant Lys (Lys72 inPKA-Ca), which has long been recognized as being essen-tial for maximal enzyme activity. This Lys lies within 13-strand 3 of the small lobe, and helps anchor and orientATP by interacting with the a- and 13- phosphates. Inaddition, Lys72 forms a salt bridge with the carboxylgroup of the nearly invariant Glu9l in subdomain III.A1a70 contributes to the hydrophobic adenine ringpocket. In PKA-Ca, 13-strand S is followed immediatelyby a-helix B, which, judging from the sequence align-ment, appears to be quite a variable structure among theprotein kinases. Indeed, this a- helix is absent in theCdk2 and Erk2 crystal structures.

Subdomain III represents the large a- helix C in thesmall lobe. The nearly invariant Glu residue (Glu9l inPKA-Ca) is centrally located in this helix and helps stabi-lize the interactions between Lys72 and the a- and 13-phosphates of ATP. Subdomain W corresponds to thehydrophobic 13-strand 4 in the small lobe. This subdo-main contains no invariant or nearly invariant residues

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Figure 2. Ribbon diagram of the catalytic core of PKAa (residues40-300) in a ternary complex with MgATP and pseudosubstratepeptide inhibitor (PKI -5-24). Invariant or nearly-invariant resi-

dues (G1y50, G1y52, G1y55, Lys72, G1u91, Aspl66, Asnl7l,Asp 184, Glu208, Asp220, and Arg280) are indicated by dots alongthe ribbon diagram. Side chains are shown for Lys72, AsplG6,Asnl7l, Aspl84, Glu208, and A.rg280. 13-strands and a-helices

are indicated by flat arrow and helices, respectively, and arenumbered according to Knighton et al. (26). The small arrow

indicates the site of phosphotransfer with the Ala in PKI substi-tuting for the phosphoacceptor Ser in the true substrate. (Repro-duced, with permission, from Taylor et al. (36)).

and does not appear to be directly involved in catalysis orsubstrate recognition.

Subdomain V links the small and large lobes of thecatalytic subunit and consists of the very hydrophobic13-strand 5 in the small lobe, the small a-helix D in thelarge lobe, and an extended chain that connects them.Three residues in the connecting chain of PKA-Ca,Glul2l, Va1123, and Glu127 help anchor ATP by forminghydrogen bonds with either the adenine or the ribosering. Metl2O, Tyr122, and Val123 contribute to the hy-drophobic pocket surrounding the adenine ring. Glu 127also participates in peptide binding by forming an ionpair with an Arg in the pseudosubstrate site of the PKAinhibitor peptide. This represents the first Arg in the PKAsubstrate recognition consensus Arg_Arg_x_Ser*_Hydrophobic.

Subdomain VIA folds into the large hydrophobic a-he-lix E that extends through the large lobe. None of the

residues in helix E appear to interact directly with eitherMgATP or peptide substrate; hence this part of the mole-cule appears to act mainly as a support structure. Subdo-main VIB folds into the small hydrophobic 13-strands 6and 7 with an intervening loop. Included here are twoinvariant residues (Asp166 and Asnl7l in PKA-Ca) thatlie within the consensus motif His-Arg-Asp-Leu-Lys-x-x-Asn (HRDLKxxN). The loop has been termed thecatalytic loop because Asp166 within the loop has.emerged as the likely candidate for the catalytic base,accepting the proton from the attacking substrate hy-droxyl group during an in- line phosphotransfer mecha-nism. Lys168 in the loop (substituted by Arg in theconventional protein-tyrosine kinases) may help facilitatephosphotransfer by neutralizing the negative charge ofthe ‘i-phosphate during transfer. The side chain ofAsn 171 helps to stabilize the catalytic loop through hydro-gen bonding to the backbone carbonyl of Asp 166 andalso acts to chelate the secondary Mg2 ion that bridgesthe a- and ‘i-phosphates of the ATP. The carbonyl groupof Glu 170 forms a hydrogen bond with an ATP ribosehydroxyl group. Glu 170 also participates in substratebinding by forming an ion pair with the second arginineof the peptide recognition consensus.

Subdomain VII folds into a 13-strand-loop-b-strandstructure, encompassing 13-strands 8 and 9. The highlyconserved DFG triplet, corresponding to Asp 184-Phe185-G1y186 in PKA-Ca, lies in the loop that is stabi-lized by a hydrogen bond between Asp184 and G1y186.Asp 184 chelates the primary activating Mg2’ ions thatbridge the 13-and ‘i-phosphates of the ATP, and therebyhelps to orient the ‘yphosphate for transfer. In Cdk2,13-strand 9 is replaced with a small a-helix designatedaLl2. However, it is unclear whether this helical charac-ter is maintained when Cdk2 is in its active conformation.

Subdomain VIII, which includes the highly conservedAla-Pro-Glu (‘APE’) motif (residues 206-208 inPKA-Ca), folds into a tortuous chain that faces the cleft.Residues lying 7-10 positions immediately upstream ofthe APE motif are characteristically well-conservedamong the members of different protein kinase families.The nearly invariant Glu corresponding to PKA-CaG1u208 forms an ion pair with an invariant Arg (Arg280in PKA-Ca) in subdomain XI, thereby helping to stabilizethe large lobe.

Subdomain VIII appears to play a major role in recog-nition of peptide substrates. Several PKA-Ca subdomainVIII residues participate in binding the pseudosubstrateinhibitor peptide. Leu198, Cys199, Pro202, and Leu205of PKA-Ca provide a hydrophobic pocket that accommo-dates the side chain of the hydrophobic residue at posi-tion +1 of the substrate consensus (Ile for the inhibitorpeptide). Gly200 forms a hydrogen bond with the samelie residue. Glu203 forms two ion pairs with the Arg inthe high-affinity binding region of the inhibitor peptide.

Many protein kinases are known to be activated byphosphorylation of residues in subdomain VIII. InPKA-Ca, maximal kinase activity requires phosphoryla-tion of Thr197, probably occurring through an intermo-lecular autophosphorylation mechanism (39). In thecrystal structure, phosphate oxygens of phospho-Thr197form hydrogen bonds with the charged side chains ofArg165, Lys189, and the hydroxyl group of Thr195, andthereby may act to stabilize the subdomain VIII loop in

an active conformation permitting proper orientation ofthe substrate peptide. For members of the Erk (MAP)kinase family, phosphorylation of both a Thr and a Tyr

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residue in subdomain VIII (mediated by members of theMEK kinase family) is required for activation. In the crys-tal structure determined for Erk2, these residues (Thr183and Tyr185) were not phosphorylated and thus the en-zyme was in an inactive state (unlike the PKA-Ca struc-ture). The unphosphorylated Tyr185 is buried in ahydrophobic pocket, and interactions with Tyr185 areapparently required to hold the enzyme in the inactivestate. Mutation of Tyr 185, however, does not activate theenzyme, and so phosphorylation of Tyr185 must also playa role in activation. Unphosphorylated Erk2 appears to beinactive because residues required for catalysis are notproperly oriented, and because its conformation resultsin a partial steric block to substrate binding. During acti-vation of Erk2, Tyr185 phosphosylation precedes Thr183phosphorylation; therefore, binding of MEK to Erk2 mayalter the conformation of the subdomain VIII loop,thereby exposing Tyr185 for phosphorylation by MEK.Interaction of phospho-Tyr185 with surface residueswould then allow the subdomain VIII loop to adopt theactive conformation (30). Subsequent phosphorylation ofthe exposed Thr 183 may activate the enzyme fully bypromoting correct alignment of the catalytic residues.From the crystal structure of Cdk2, likewise in an inactiveunphosphorylated state, the subdomain VIII loop appearsto be in a conformation that would inhibit enzyme activityby sterically blocking the presumed protein substratebinding cleft (29). Phosphorylation of Thr160 in the Cdk2subdomain VIII, mediated by M015 (CAK), presumablywould act to remove this inhibition by stabilizing the loopin an active conformation similar to that found inPKA-Ca. Cyclin binding to the NH2-terminal lobe is alsoneeded to activate Cdk2, and this may cause rotation ofthe NH2-terminal domain resulting in correct alignmentof catalytic residues.

Subdomain IX corresponds to the large a- helix F ofthe large lobe. The nearly invariant Asp corresponding toPKA-Ca Asp220 lies in the NH2-terminal region of thishelix and acts to stabilize the catalytic loop by hydrogenbonding to the backbone amides of Arg165 and Tyr164that precede the loop. G1u230 of PKA-Ca forms an ionpair with the second Arg of the peptide recognition con-sensus. PKA-Ca residues 235-239 are all involved in hy-drophobic interactions with the inhibitor peptide.

Subdomain X is the most poorly conserved subdomainand its function is obscure. In the crystal structure ofPKA-Ca, it corresponds to the small a-helix G that occu-pies the base of the large lobe. Members of the Cdk, Erk(MAP), GSK3, and Clk kinase families (the C-M-G-Cgroup) all have rather large insertions between subdo-mains X and XI, whose functional significance is presentlyunclear. Subdomain XI extends to the COOH-terminalend of the kinase domain. The most notable feature hereis the nearly invariant Arg corresponding to Arg280 inPKA-Ca, which lies between a-helices H and I. TheCOOH-terminal boundary of the kinase domain is stillpoorly defined. For many protein-serine kinases, the con-sensus motif His-x-Aromatic-Hydrophobic is found be-ginning 9- 13 residues downstream of the invariant Arg.For protein-tyrosine kinases, a hydrophobic amino acidlying 10 positions downstream of the invariant Arg ap-pears to define the COOH-terminal boundary.

The amphipathic a-helix A of PKA-Ca (residues15-35; not shown in Fig. 2), though lying outside of theconserved catalytic core on the NH2-terminal side, ap-pears to be an important feature found in many protein

kinases (40). This helix spans the surface of both lobes ofthe core structure and complements and stabilizes thehydrophobic cleft between the two lobes. The A-helixmotif appears to be present in many other protein kinasesincluding members of the protein kinase C family and theSrc family of protein-tyrosine kinases (40).

CLASSIFICATION OF EUKARYOTIC PROTEINKINASES

To facilitate analysis and management of this large super-family we have devised the classification scheme shown inTable 1, which subdivides the known members of theeukaryotic protein kinase superfamily into distinct fami-lies that share basic structural and functional properties.Phylogenetic trees derived from an alignment of kinasedomain amino acid sequences (essentially an expandedversion of Fig. 1) served as the basis for this classification.Thus, the sole consideration was similarity in kinase do-main amino acid sequence. When considered alone, how-ever, this property has been a good indicator of othercharacteristics held in common by the different membersof the family.

Protein kinases whose entire kinase domain amino acidsequence had been published by July 1993 were includedin phylogenetic analysis (as well as a few others madeavailable at that time through sequence databases). If agiven kinase domain sequence had been determined frommore than one species among the vertebrates (i.e., or-thologous gene products), only one representative (usu-ally human) was included in the analysis. This policy wasnot used for the other phyla, however, because of greaterdivergences between the species and, hence, the se-quences. The kinase domain phylogenies were inferredusing the principle of maximum parsimony according tothe PAUP software package developed by Swofford (41).Minimum-length trees were found using PAUP’s ‘heuris-tic’ search method with branch swapping by the ‘treebisection-reconnection’ strategy. Equal weights weregiven for all amino acid substitutions. Because multipleminimum-length trees were found, a consensus tree wascalculated according to the method of Adams (cited in ref41) in order to show branching ambiguities.

To accommodate the large numbers of sequences, itwas necessary to construct five separate trees. Initially, askeleton tree of 99 kinases was obtained (Fig. SA). Theskeleton tree included only representative members fromeach of four large groups of protein kinases, each consist-ing of multiple related families known from previouswork to cluster together in the tree. These four groupsare designated: 1) the AGC group, which includes thecyclic-nucleotide-dependent family (PKA and PKfL), theprotein kinase C (PK) family, the 13-adrenergic receptorkinase (13ARK) family, the ribosomal S6 kinase family, andother close relatives; 2) the CaMK group, which includesthe family of protein kinases regulated by calcium/cal-modulin, the Snfl/AMPK family, and other close rela-tives; 3) the CMGC group, which includes the family ofcyclin-dependent kinases, the Erk (MAP) kinase family,the glycogen synthase 3 (GSK3) family, the casein kinaseII family, the Clk (Cdk-like kinase) family, and other closerelatives; and 4) the ‘conventional’ protein-tyrosine ki-nase (PTh) group. Separate trees (Fig. 3B-E) were laterobtained for each of the four large kinase groups, andcontain all members of the groups whose sequences wereavailable at the time of analysis.

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Ccth15Cin16

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E

It can be reasonably surmised that the protein kinaseshaving closely related catalytic domains, and thus defininga family, represent products of genes that have under-gone relatively recent evolutionary separations. Giventhis, it should come as no surprise that members of agiven family tend also to share related functions. This ismanifest by similarities in overall structural topology,mode of regulation, and substrate specificity. The detailsof the common properties exhibited by the members ofthe various kinase families can best be gleaned fromstudying the information outlined in the individual en-tries section of the Protein Kinase Factsbook (42). Some ofthe most salient relationships are discussed below.

The AGC group protein kinases tend to be basic aminoacid-directed enzymes, phosphorylating substrates atSer/Thr residues lying very near Arg and Lys. For thecyclic nucleotide-dependent and ribosomal S6 kinasefamilies, the preferred substrates have basic residues lyingin specific positions NH2-terminal to the phosphate ac-ceptor. Preferred substrates for the PKC and RAC fami-lies have basic residues on both the NH2- and COOH-terminal sides of the acceptor (43). The C-protein-cou-pled receptor kinases (13ARK and RhK) appear to breakthis rule, however, as they are reported to prefer syntheticpeptide substrate residues located within an acidic envi-ronment. Little substrate information is available for theother families in this group.

Figure 3. Phylogenetic trees of the eukaiyotic protein kinasesuperfamily inferred from kinase domain amino acid sequencealignments. The abbreviated nomenclature is the same used inTable 1. A) ‘Skeleton’ tree showing 99 protein kinases. Positionsof 4 clusters (AGC, CaMK, CMGC, and PTK) containing proteinkinases representative of larger groups are indicated in the skele-ton tree. B) AGC group tree of 59 protein kinases including PKA,PKG, and PKC and other close relatives. C) CaMK group tree of35 protein kinases including the calcium/calmodulin-regulatedenzymes. D) CMGC group tree of 59 protein kinases includingthe cyclin-dependent kinases. E) PTK group tree of 90 conven-tional protein-tyrosine kinases. Tree A is unrooted and drawnwith Pknl and Pkn2 as outgroups. Outgroups of two or moredistantly related protein kinases (not shown) were included in theanalysis of trees B-E to provide a rooting point. Asterisks (*) inall trees indicate branches leading to defined protein kinasefamilies listed in Table 1. Branch lengths indicate number ofamino acid substitutions required to reach hypothetical commonancestors at internal nodes.

The CaMK group protein kinases also tend to be basicamino acid- directed, and in this regard it is notable thatthe AGC and CaMK groups fall near one another in thephylogenetic tree. CaMK1, CaMK2, CaMK4, MLCK,CDPK, and AMPK are all reported to prefer substrateswith basic residues at specific positions NH2-terminal tothe acceptor site, whereas EF2K and PhK prefer sites withbasic residues at both NH2- and COOH-terminal loca-tions. Many, but not all, of the CaMK group proteinkinases are known to be activated by Ca’7calmodulinbinding to a small domain located just COOH-terminalto the catalytic domain, e.g., CaMK1, CaMK2, CaMK4,PhK’i, MLCK, and twitchin. These enzymes and theirclose relatives are grouped together in a large familywithin the CaMK group. Also included in this family area subfamily of plant enzymes (represented by CDPK) thatcontain an intrinsic calmodulin-like domain that confersCa2-dependent activation. The other family within theCaMK group is the Snfl/AMPK family. Within this fam-ily, substrate specificity determinant information hasbeen obtained only for the AMP-activated protein kinase,which also shows a requirement for an NH2-terminalbasic residue. The other major category of protein-serinekinases is the CMGC group. For the most part, these areproline-directed enzymes, phosphorylating substrates atsites lying in Pro-rich environments. Available data forCdc2 and Cdk2 indicate that members of the cyclin-de-

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pendent kinase family require phosphate acceptors lyingimmediately NH2-terminal to a Pro. A similar require-ment is indicated for the Erk (MAP) kinase family. Thesituation for the GSK3 family is more complicated, butmost known acceptor sites lie within Pro-rich regions.The structures of Cdk2 and Erk2 indicate that the pocketfor the +1 residue is shallower than in PKA-Ca due to thereplacement of Leu205 by an Arg, which is bulkier andprecludes binding of the larger hydrophobic amino acids.In addition, the unique secondary amide group of Promay make special interactions (44). The casein-kinase IIfamily enzymes fail to conform to the proline-directedspecificity exhibited by the other major families of thisgroup, showing instead a strong preference for Ser resi-dues located NH2-terminal to a cluster of acidic residues.The CMGC group protein kinases have larger-than-aver-age kinase domains due to insertions between subdo-mains X and XI, whose functional significance isunknown.

The conventional protein-trosine kinase group in-cludes a large number of enzymes with quite closely re-lated kinase domains that specifically phosphorylate onTyr residues (i.e., they cannot phosphorylate Ser or Thr).These enzymes, first recognized among retroviral onco-proteins, have been found only in metazoan cells wherethey are widely recognized for their roles in transducinggrowth and differentiation signals. Included in this groupare more than a dozen distinct receptor families made upof membrane-spanning molecules that share similar over-all structural topologies, and nine nonreceptor familiesalso composed of structurally similar molecules. Thespecificity determinants surrounding the Tyr phosphoac-ceptor sites have yet to be firmly established for theseenzymes, but Glu residues either on the NH2- or COOH-terminal side of the acceptor are often preferred. Thisgroup is labeled “conventional” to distinguish it fromother protein kinases (including Spkl, Cik, the MEK/Ste7family members, Weel/Miki, ActRII, Hrr25, Esk, andSp1A/DPyk2) reported to exhibit a dual specificity, thatis, being capable of phosphorylating both Tyr andSer/Thr residues (45). However, in most cases dual speci-ficity has been observed only for autophosphorylationreactions in vitro, and the only dual specificity proteinkinases that are known to be able to phosphorylate asubstrate on Ser/Thr and Tyr are members of the MEKfamily. Considered as a group, these dual-specificity pro-tein kinases are not particularly closely related to theconventional PTKs. Indeed, they seem to map through-out the phylogenetic tree (45), suggesting that the abilityto autophosphorylate on Tyr may have had many inde-pendent origins during the evolutionary history of thesuperfamily.

The protein kinases falling outside the four majorgroups are a mixed bag. Although the individual mem-bers within the defined families found in this “other”category clearly are related to one another through bothstructure and function, it is difficult to make broadergeneralizations that could group any of these familiestogether into a larger category. As far as substrate speci-ficity determinants go, little is known about most “other”category protein kinases, due primarily to their ratherrecent discovery and the paucity of known physiologicalsubstrates. The casein kinase I family members, however,have been shown to prefer Ser/Thr residues locatedCOOH-terminal to a phosphoserine or phosphothreon-me, although a stretch of acidic residues may substitute.

Also, the family of protein kinases involved in transla-tional control (HRI, PKR/Tik, Gcn2) appear to be basicamino acid-directed enzymes preferring Ser residues ly-ing NH2- terminal to an Arg. Finally, as mentioned pre-viously, the MEK/Ste7 family protein kinases andWeel/Miki protein kinases exhibit a dual specificity.

Although this classification is based solely on catalyticdomain sequences, members of families defined by thismeans are usually closely related in regions lying outsidethe cataytic. domains and in many cases have been shownto possess very similar functions. Thus, intercalation ofnewly discovered protein kinases into this classificationshould allow one to make useful predictions about thefunctions of such enzymes.

FUTURE PROSPECTS

The rate of protein kinase discovery still shows no signsof abating. In addition to the continuing successes ofhomology-based approaches, genomic sequencing pro-jects are beginning to make significant contributions. Forinstance, the sequences of two entire budding yeast chro-mosomes (46, 47) and a 2 Mb stretch of C. elegans chro-mosome III (48) have revealed a number of new putativeprotein kinase genes. As genome sequencing projectsgather speed, the number of new protein kinase genesdiscovered in this way will undoubtedly mushroom. Thisexplosion of sequence data is making it increasingly diffi-cult to manage protein kinase databases of the sort de-scribed here. Programs designed to align and deriverelatedness trees are currently unable to handle the largenumber of available kinase domain sequences. New datahandling programs will have to be developed to cope withlarge numbers of sequences like those of the eukaryoticprotein kinase superfamily.

Protein kinase catalytic domain structures will continueto be solved. The first structure of a conventional pro-tein-tyrosine kinase will be available shortly (see “Noteadded in proof’), and this should reveal how Tyr is se-lected as an acceptor amino acid vs. Ser/Thr. Such struc-tures will enable comparative analysis to be carried out atthe 3-dimensional level, and allow predictions of struc-tures from primary sequences. Structural comparisons ofcatalytic domains with bound peptide substrates will alsoprovide insights into substrate specificity. Most proteinkinases show some degree of primary sequence specific-ity, and new methods are being developed to determineconsensus sequence specificities for individual protein ki-nases (44). With such consensus information the struc-tural basis for the binding of a preferred peptidesequence to the cognate substrate binding site can thenbe deduced. In the future, it may be possible to model theS-dimensional structure of a novel protein kinase cata-lytic domain with sufficient accuracy to be able to deducethe preferred primary sequence surrounding the hy-droxyamino acid it phosphorylates, which in turn willallow one to predict what proteins might be its substratesfrom the increasingly complete database of protein se-quences.

Note added in proof: The crystal structure of the tyrosine kinasedomain of the insulin receptor has now appeared (Hubbard,S. R., Wei, L., Ellis, L., and Hendrickson, W. A. (1994) Nature 372,746-754).


SERIAL REVIEW.

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kinase superfamily: (catalytic) domam structure and classification

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