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member of the SNF2 family differs from one block to another, with five different proteins represented (Fig. la). Therefore, the SNF2 family appears to have diverged fairly uniformly from a common ancestor shared with NTPI.

The detection of global homology to poxvirus DNA-dependent ATPases has probable relevance to mechanistic models for the SNF2 group proteins. One vaccinia homolog is a subunit of the early transcription factor e. Another is a DNA-dependent ATPase w~th well-studied substrate and cofactor requirements s.

Efforts to detect a helicase activity in VATP group proteins have been unsuccessfuP; therefore, serious consideration should be given to ATP-dependent roles for the SNF2 proteins that do not require unwinding, such as movement along duplex DNA. In this regard, transcription of vaccinia virus in autonomous cytoplasmic particles provides a relatively simple model .'hat is _lot hampered by the poorly defined nuclear and chromatin environment in which the proposed nuclear complexes normally operate.

References 1 Travers, A. A. (1992) Cell 69, 573-575 2 Hemkoff, S. and Hentkoff, J. G. (1991) Nucleic

Acids Res. 19, 6565-6572 3 Koonm, E. V, (1991) Nature 352, 290 4 Broyles, S, S. and Fesler, B S. (1990) J. Wrol,

64, 1523-1529 5 Paolettl, E, and Moss, B. (1974) J, BfoL Chem.

10, 3281-3286 6 Kunn, M, S, and Traktman. P (1989) J. V~rol.

63, 3999-4010

STEVEN HENIKOFF

Howard Hughes Medical Institute, Fred Hutchinson Cancer Research Center, Seattle, WA 98104, USA.

Sequence similarity of phospholipase A= activating protein and the G protein subunits: a new concept of effector protein activation in signal transduction?

Phospholipase A 2 (PLA~) activation has been implicated as a key factor in the release of arach~donic acid and the subsequent production of oxygenated eicosanold metabolites. These metabolites govern cellular functions as diverse as inflammation, natural killer activities, smooth muscle contraction, mn channel activities, neurotransmission and wsual signal transduction ~. There is increasing ewdence that heterotrimeric G proteins mediate the stimulation of PLAz in many cell types, including neutrophils,

fibroblasts, sensory neurons and retinal rods z

Activation of trimenc G proteins involves their dissocmtion into o~- and I]7-subunits, thereby generating two potential regulatory molecules 3. Based on the well-documented stimulation of adenylate cyclase, phospholipase C and cGMP phosphodiesterase by G. it was initially believed that the ¢x-subunit is the active component mediating PLA z stimulation Recently, however, a few examples of direct effects of 157-subunits

(a ) (b ) ' / / PLAP 48 - LYIL~3"-I~TVCSLSSGKFGT'LLSGS;WI~TAK~ -L-ND! - 86 .¢:: 2001

PLAP 127 ERTFI~G F~DCVRGLAI LSETE - FLS~ Z P~ 3Z-TG~ 166 PLAP 167 LEV'~]GIITNYIySISVFPNSKDFVTTAE~LgJ E'-HG| 206 ~ 1

~-Trans 84 TTTP~G~TGDVMSLSLAPDTRLFVSGAC~ASAK~ )VREG~ 125 GBLP 99 TRRF~G~TKD ~LSVAF SVDNRO IVSGS R~T IKl .~TL-G| 139 SFL2 343 VMI L~G~EQD Z YSLDYFP SGDKLVSGSG~KTVR/ )LKTG( 475 ;~c3 ~zz ',KT'-~F~,SZYC~J'D~'TGK~GS~SZVSZ ~ZED~ 352 CDC4 415 LLQL~G~GGVWALKYAHGG-ILVSGST~TVR~ )Z-KK( 454 2'0" " 'I00 '' 200''' 300" PRP4 256 LGDL~G~RRZ SDVK~HP SGKFXGSASH~T~ DASTHQE 299 PLAP STE4 174 AS I F~G~TCY I SD ZEFTDNAHX L-TAS~TCA~ )~9KAKR 214 Ycu7 z94 LO~FGPVSCUSFSOm~SW.aS~,S',9~Z~U SZrGRSO 3~ (¢) MAKI1 196 VGTL~G~TA~VNDVDZ HP TNRIAZSVSD~$ IR~ ~LMTLRN 233 GROU 426 Q I NT ~S ~GEVVCAVT~ SNPTKYV~- TGG~GCI/K% DTSQPGN 466 COP1 542 LHVF ~G ~KKAVS ¥MKF LSNNEL-ASAS T~STLRI DVKDNLP 582

coxs ~ pH~ v ~$~$ p~o'J:#l~sGs,~ ' ~ z :~ ~.c PLAP I I t li ~ . , , , i ® l I _ _ 1 Z ~r 000' El o" 4 ¢ mc p-Trans I[q-II e l l S I 4 a--~'i S " 7 I al i

I I I I

0 100 200 300 Residues

Rgme 1 (a) Sequence alignment of the four tandem repeats of PLAP with a typical repetitive segment of human ~-transducin (GenPept:HUMTRNBI_I) and other members of the superfamily: GBLP (SwlssProt:GBLP_HUMAN), human guanine nucleotlde-binding protein [5 12.3, product of a human MHC locus gene; SFL2 (pir:S11169), yeast flocculatlon suppressor protein SFL2; AAC3 (SwissProt:AAC3_DICDI), AAC-rlch mRNA from Dzctyostelhum dfscozdeum; CDC4 (SwissProt:CC4_YEAST), yeast celldivtsion control protein 4; PRP4 (SwlssProt:PRO4_YEAST), yeast U4/U6 small nuclear nbonucleoprotem; STE4(GenPept:YSCSTE4_I), yeast mating factor receptor-coupled G protein; YCU7 (SwlssProt:YCU7_YEAST), yeast hypothetical guanine nucleotide-bindlng protein [5; MAK11 (SwissProt:MK11_YEAST), membrane-associated yeast MAK11 protein required for the maintenance of killer M1 double-stranded RNA; GROU (Sw,ssProt:GROU_DROME), Drosophila melanogaster groucho protein or enhancer of Spl~, product of a neurogen¢ gene; COP1 (not yet submitted to databases), an Arabidopsls regulatory gene containing a zinc- binding motif 13. Residues identcal in 80% of the repeats and the carboxy-termmal cysteme found m several repeats are boxed; tinted areas indicate positrons where at least 70% of the residues have similar properties; (+/_+, charged; ¢, hydrophobic; o, small polar; ~, polar). CONS, a consensus sequence where residues are indcated if at least 50% are ident~cal at a g~ven pos~hon. (b) Diagonals of homology with scores above 50 (based on the PAM250 scoring matrix) between mouse PLAP (SwissProt: PIAP_MOUSE) and human ~transducin {Genbank:HUMTRNB1) using PLFASTA. (c) Schematic representation of PLAP and ~-transducm. Numbered boxes correspond to the tandem repeats, M represents the domain having sequence slmdanty with melhtm.

292 © 1993. Elsevier Science Publishers, LqJIQ 0968-0004/93/$06.00

TIBS 1 8 - A U G U S T 1 9 9 3

have been reported. The targeting of the [$-adrenergic receptor Idnase to membrane-bound receptors, leading to receptor desensitization, is mediated by [3y-subunits 4. G protein ~f-subunit- mediated regulation of signal pathways including phospholipase C (Ref. 5) and adenylate cyclase 6 were also reported. The stimulation of PLA2 activity by GI~ was observed in the rod photoreceptor outer segment of the retina ~. Gpy or 137.1ike proteins were therefore predicted to be stimulatory to PLA 2

It is well known that bee venom mellitin is an efficient stimulator of PLA z. In an attempt to find a mammahan counterpart of the bee venom peptide, the cDNA of a PLA 2 activating protein (FLAP) was cloned by screening an expression library with antibodies to mellitin B. The carboxy-terminal portion o! the PLAP sequence reveals a striking similarity to melhtm. Synthetic ant[sense PLAP DNA was shown to block the activation of PLA2 and subsequent production of eicosanoids, implying a crucial role for PLAP in PLA z activation. Moreover, a synthetic peptide spanning the PLAP-melhtin homology sequence led to the stimulation of PLA2, providing direct evidence for its effector role s. However, no homology of PLAP to G proteins has been reported.

We have recently characterized a novel organelle-associated protein with sequence similarity to the G protein ~-subunit (D. Masson et al., unpublished). While searching the NCBI nonredundant protein database for slmllaritles with other proteins using the BLAST program g, we Identified PLAP as a new member of the ~transducln (GI0 superlamlly. The yeast flocculatlon suppressor protein SFL2 ~° was found to be the protein related most closely to PLAP, with chance probabilities of I,Ixl0 "~.

To date, the p-transducin superfamily comprises more than l l nonorthologous proteins =l']z. Only two distinct ~subunits are described in mammals(four distinct ~transducin isotypes, GI~ =-4 and the guanine nucleotide-binding protein 12.3), whereas all others occur primarily in yeast. The known members of this superfamily contain between five and nine internal '~-transducin' repeats containing approximately 40 amino acids. lS-Transducin consists of eight repeats spanning the complete protein but PLAP contains only four such modules covering residues 48-206 (Fig. la). A hallmark of these repeats is a tryptophan punctuation predominantly followed by an aspartate (~transducin repeats are also called WD-repeats). The four PLAP repeats show either Lys, Gin or Leu instead of an aspartate at this positron (Fig. la). Most other members of this superfamily contain at least one repeat with a mutated WD dipeptide, where the aspartate is replaced by either Asn, Gin, Arg, Lys or Ser. All other features of the repeats are conserved (Fig. |a) and thus allow their classification as genuine but more distantly related ~-transducin domains. The homology between PLAP and ~transducin thus strengthens the notion that G proteins play a key role in the activation of PLA 2.

Interestingly, the ~transducin homology region of PLAP is directly fused to the mellitin-containing carboxy- terminal effector domain. Thus, a new concept of effector-protein activation in signal transductlon Is emerging. Unlike the classical Gl~-Subunlts that form a noncova|ent complex wi th the ?-subunit, this Gp is d i rect ly l inked to an effector moiety. We can envisage a new G o family (G{~e~t) in which the G o homology region is fused to various domains, each having a

distinct effector function possibly regulated by components of the G protein signalling pathway, indeed, our recently identified murine GI3 homoiogue ]s contiguous with a 100 Id)a carboxy- terminal extension, and most of the Gp superfamlly members in yeast bear (an) extra amino-terminal domain(s) with as yet poorly characterized functions. Since some of these Gp proteins are localized either in the cytoplasm or the nucleus, their site of action may not be limited to the plasma membrane.

References J. Pruzansk=, W and Vadas, P (1991) Immunol

Today 12, 143-146 2 Axelrod, J,, Butch, R. M, and Jelsema, C L

(1988) Trends Neurosc~ 11, 117-123 3 Simon, M. I,, Strathmann, M P and Gautam, N

(1991) Science 252, 802-808 4 P~tcher, J. A, et al (1992) Science 257,

1264-1267 5 Camps, M, et al. (1992) Eur. J. BJochern 260,

821-831 6 Tang, W J and Gilman, A G, (1991) Science

254, 1500-1503 7 Jelsema, C L. and Axelrod, J (1987) Proc, Nat/

Acad Sct. USA 84, 3623-3627 8 Clark, M. A et aL (1991) Proc. Natl Acad Sc~

USA 88, 5418-5422 9 Altschul, S. F, et aL (1990) J Mot Bzo/ 215,

403-410 10 Fujlta, A. et al {1990) Gene 89, 93-99 11 Duronm, R J., Gordon, J. I and Bogusk~, M. S

(1992) Proteins 13, 41-56 12 van der Voom, L. and Ploegh, H L. (1992) FEBS

Lett. 307,131-134 13 Deng, X. W. et al. (1992) Cell 71, 791-801

MANUEL C. PEITSCH, CHRISTOPH BORNER AND J(IRG TSCHOPP

Insbtute of Biochemistry, University of Lausanne, CH.1066 Epahnges, SwLtzerland

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