© 1999 Macmillan Magazines Ltd

news and views

NATURE CELL BIOLOGY | VOL 1 | AUGUST 1999 | cellbio.nature.com E93

part in regulating cell-cycle entry and exit5,6, wewill need to understand whether SCFSKP2-mediated degradation of p27 is directly regu-lated by extracellular stimuli, or is a secondary(but necessary) consequence of G1 progres-sion and CDK2 activation. Likewise, we still donot know whether SCFSKP2 interacts function-ally with other proteins that control p27 func-tion, such as c-Myc, D-type cyclins, Ras5–7,Jab1 (ref. 14) and possibly tumour suppressorssuch as VHL, PTEN and Tsc-2 (for example,ref. 7). New exciting connections are likely to

emerge in the near future. hBruno Amati and Jaromir Vlach are at the Swiss Institute for Experimental Cancer Research, CH-1066 Epalinges, Switzerland.e-mail: bruno.amati@isrec.unil.ch

1. Koepp, D. M., Harper, J. W. & Elledge, S. J. Cell 97, 431–434 (1999).

2. Harper, J. W. & Elledge, S. J. Nature Cell Biol. 1, E5–E7 (1999).

3. Carrano, A. C., Eytan, E., Hershko, A. & Pagano, M. Nature Cell

Biol. 1, 193–199 (1999).

4. Sutterlüty, H. et al. Nature Cell Biol. 1, 207–214 (1999).

5. Sherr, C. J. & Roberts, J. M. Genes Dev 13, 1501–1512 (1999).

6. Amati, B., Alevizopoulos, K. & Vlach, J. Front. Biosci. 3, D250–

D268 (1998).

7. Clurman, B. E. & Porter, P. Proc. Natl Acad. Sci. USA 95, 15158–

15160 (1998).

8. Tsvetkov, L. M., Yeh, K. H., Lee, S. J., Sun, H. & Zhang, H. Curr.

Biol. 9, 661–664 (1999).

9. Lisztwan, J. et al. EMBO J. 17, 368–383 (1998).

10. Zhang, H., Kobayashi, R., Galaktionov, K. & Beach, D. Cell 82,

915–925 (1995).

11. Dyson, N. Genes Dev. 12, 2245–2262 (1998).

12. Marti, A., Wirbelauer, C., Scheffner, M. & Krek, W. Nature Cell

Biol. 1, 14–19 (1999).

13. Zhang, H. S., Postigo, A. A. & Dean, D. C. Cell 97, 53–61

(1999).

14. Tomoda, K., Kubota, Y. & Kato, J. Nature 398, 160–165

(1999).

Calcium – myristoyl switches turn on new lights

Tobias Meyer and John D. York

The yeast homologue of the calcium–myristoyl-switch protein frequenin activates a key enzyme in the phosphoinositide signalling cascade, shedding new light on the role of such switch proteins and of calcium signalling.

alcium–myristoyl-switch proteins con-stitute a new subfamily of ‘EF-hand’calcium-ion sensors, so named because

their Ca2+-binding domains consist of a motifknown as the EF-hand. These proteins haveattracted attracted much attention as theycontrol synaptic activity as well as recoveryand adaptation in visual signal transduction.Members of this family include recoverin,GCAP, frequenin and many others. In thevisual response of vertebrate rod outer seg-ments, recoverin and GCAP are thought tofunction in the dark, in high cytosolic Ca2+

concentrations, as inhibitors of the enzymesrhodopsin kinase and guanylate cyclase1,respectively; light-induced lowering ofcytosolic Ca2+ allows these enzymes to beactivated. Drosophila mutants that overex-press frequenin at the neuromuscular junc-

tion exhibit a striking facilitation ofneurotransmitter release, indicating a linkbetween activation of frequenin by Ca2+ andregulated secretion2. And on page 234 of thisissue, Hendricks et al.3 suggest a new target ofCa2+ signalling by showing that a yeast homo-logue of frequenin acts as an activator ofphosphatidylinositol-4-OH kinase, a keyenzyme in the phosphoinositide signallingsystem.

The frequenin and recoverin subfamily ofEF-hand signalling proteins is characterizedby an intriguing activation process that hasbeen termed a Ca2+–myristoyl switch4,5. Thenuclear magnetic resonance structure ofmyristoylated recoverin in its Ca2+-free stateshowed that the myristoyl group, a fatty-acidtag, is sequestered in a hydrophobic cavity ofthe protein that is lined with many aromatic

C

and hydrophobic residues6. This cavity wasseen in the myristoyl-free X-ray structure as ahydrophobic groove7. The binding of twoCa2+ ions to recoverin leads to the extrusionof the fatty acid, making it available to inter-act with membrane targets8. The extrusion ofthe fatty acid is also accompanied by a largeconformational change in the protein, caus-ing exposure of the hydrophobic groove. Theexposed amino acids may serve as a bindingsite that suppresses or activates potentialbinding partners. Many of the hydrophobicresidues that interact with the myristoylgroup are highly conserved in frequenin andhomologues from yeast to humans, so theCa2+–myristoyl-switch mechanism may besimilar for all proteins of this subfamily.These structural data leave open two possiblemechanisms by which Ca2+–myristoyl-switchproteins can activate their targets (Fig. 1). Foreach protein, these mechanisms may func-tion separately or synergistically.

What is the significance of Hendricks etal.’s finding3, that yeast frequenin (Frq1) acti-vates phosphatidylinositol (PtdIns)-4-OHkinase? The regulation of phosphoinositidesis crucial to cellular function. These lipidsform an emerging family of membrane-spe-cific second messengers that control mem-brane trafficking, organize cytoskeletalstructures, and regulate signalling proteins9.In this context, phosphoinositides such asPtdIns-4-phosphate (PtdIns(4)P) andPtdIns-4,5-bisphosphate (PtdIns(4,5)P2)can be seen as members of an ensemble of

Figure 1 Two mechanisms by which Ca2+–myristoyl-switch proteins may activate their targets. In the translocation model, Ca2+ binding triggers the translocation of the switch protein and its enzyme target to the membrane. In the activation model, the Ca2+–myristoyl-switch protein is already prelocalized to a membrane and the Ca2+-induced conformational change triggers the activation of membrane-localized targets. Extrusion of the switch protein’s myristoyl tag upon binding of Ca2+ enables the protein to interact with membrane targets. Mixed processes, in which either the Ca2+ sensor or the target translocates to the membrane, may also occur for some members of the Ca2+–myristoyl-switch family of proteins.

Target

TargetSwitchprotein

Activetarget

Plasmamembrane

Myristoyl tag

Ca2+-mediated

Ca2+-mediated

activation model

translocation model

© 1999 Macmillan Magazines Ltd

news and views

E94 NATURE CELL BIOLOGY | VOL 1 | AUGUST 1999 | cellbio.nature.com

coloured ‘lights’ that project onto variouscellular membranes or ‘stages’. An ability toalter the intensity and composition of theensemble allows the cell to rapidly changestage sets, which may be interpreted by cellu-lar machinery to elict specific signallingresponses (Fig. 2). The switchboard that con-trols the lights consists of numerous genesencoding lipase, kinase and phosphataseactivities that transiently remodel the phos-phatidylinositol composition of individualmembranes. One can quickly grasp the com-binatorial complexity that can be achieved indifferent types of membranes by mixing alimited number of ‘primary-colour’ PtdInssignalling molecules.

PtdIns(4)P, one of the essential lipids inthe pathway that generates different types ofphosphoinositide, is synthesized from PtdInsthrough phosphorylation at the D-4 positionof the inositol ring by PtdIns-4-OH kinases10.PtdIns(4)P can be metabolized throughthree activities: first, dephosphorylation bythe 4′-phosphatase activity of proteins con-taining so-called SAC1-like domains11; sec-ond, hydrolysis by phospholipase C12; andthird, further phosphorylation of the D-3 orD-5 ring position by 3′ and 5′ kinases,respectively10. The balance of these and otherenzyme activities defines a specific PtdIns‘colour’ for each cellular ‘stage’.

What are the specific roles of PtdIns(4)Pand PtdIns(4,5)P2? An important role ofPtdIns(4)P is that of a precursor, enablingproduction of PtdIns(4,5)P2 as well as thesecond messengers inositol-1,4,5-trisphos-phate (InsP3), diacylglycerol and PtdIns-3,4-bisphosphate. Numerous studies havedocumented a signalling role forPtdIns(4,5)P2. The regulatory mechanisms,subcellular localizations and binding part-ners of these lipids are now being eluci-dated. Although many groups haveinvestigated the role of PtdIns(4)P as a pre-cursor to the later second messengers, fewhave attempted to understand a possibledirect signalling function for this lipid.

Generating results that impinge on bothof these strands of research — study of thefunctions of Ca2+–myristoyl-switch proteinsand of the role of phosphoinositides — Hen-dricks et al.3 use a genetic approach to iden-tify targets of yeast Frq1, and find that acritical target is one of the two known yeastPtdIns-4-OH kinases, Pik1. This surprisingresult links the sole yeast Ca2+–myristoyl-switch protein to the regulation of a subcel-lular phosphoinositide pool(s).

The biochemistry of Frq1 and Pik1 inter-action shows that Frq1 is indeed myris-toylated and that the Ca2+-bound proteinfunctions as an activator of Pik1’s lipid-kinase

activity. This suggests a new link betweenyeast Ca2+ signals and the regulation ofPtdIns(4)P synthesis. The genetics of thisstudy provides provocative insight into therole of PtdIns(4)P as a possible cellular signal-ling molecule. The demonstration that Pik1but not the other essential yeast PtdIns-4-OHkinase — Sst4 — can rescue a frequenindefect, the essential nature of both Pik1 andStt4, and the inability of either enzyme tocompensate for the other, even when overex-pressed, indicates that unique PtdIns(4)Plipid pools exist in the cell that may be inter-preted as distinct signals or colours (Fig. 2).

It is also intriguing that the genetic screenin this study failed to find either the yeastPtdIns(4)P-5-OH kinase (Mss4) or the yeastphospholipase C (Plc1). This is significant, asoverproduction of Mss4 can rescue a stt4mutant13. It has also been shown that, in nor-mal yeast cells, overproduction of Mss4 andPlc1 results in increased levels ofPtdIns(4,5)P2 and InsP3, respectively14,15.Thus, it appears that an increase in cellularPtdIns(4,5)P2 and/or InsP3 is not sufficientto overcome loss of frequenin activation ofPik1. These results leave open the possibilitythat frequenin drives production of a specificPtdIns(4)P pool that may function as a directsecond messenger or as a precursor to aunique essential pool of PtdIns(4,5)P2 orother downstream molecules.

Can Hendricks et al.’s results3 be used togain insight into the cellular role of frequeninin other cell types? Given the direct relation-ship of frequenin and phosphoinositides inyeast, the connection between frequenin andneurotransmitter secretion in Drosophilaand the established roles of phosphoi-nositides in secretion, we speculate that fre-quenin functions as a regulator of exocytosisand membrane trafficking through the acti-vation of phosphoinositide signalling. Thisraises new questions. Is the main role of Ca2+-activated frequenin to direct membrane traf-ficking through PtdIns(4)P? At which sub-cellular membranes does activation offrequenin and Pik1 occur? On the upstreamside of frequenin, the paper raises the inter-esting question of how Ca2+ signals can begenerated that regulate yeast frequenin. Ca2+

regulation in yeast is thought to be differentfrom the canonical model of mammaliancells, as no genes encoding InsP3-receptorhomologues, the activation of which isinvolved in Ca2+ release from intracellularstores, have been identified in the yeastgenome. Thus, searching for upstreammechanisms of activation of yeast frequeninmay give interesting new insights into Ca2+

regulation in yeast and other cell types. hTobias Meyer is in the Departments of Cell Biology, and Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.John D. York is in the Departments of Pharmacology and Cancer Biology, and Biochemistry, Duke University Medical Center,

Figure 2 The hand of Ca2+-bound frequenin directs changes in phosphoinositide signals. The signalling phenotype is seen as a cellular ‘stage’ illuminated by selected phospho-inositide ‘lights’, such as phosphatidylinositol (PtdIns)-4-phosphate (PtdIns(4)P; yellow), PtdIns-4,5-bisphosphate (PtdIns(4,5)P2; red) and PtdIns-3,4-bisphosphate (PtdIns(3,4)P2; cyan). Switches represent enzymes that regulate production of these molecules. The PtdIns-4-OH kinases, Pik1 and Stt4, and PtdIns(4)P kinases are labelled. Sliding switches represent other phosphatase and lipase activities that also participate in controlling lipid levels. The circuitry enabling the flow of ‘electricity’ to generate individual reactions is shown by plugs and sockets. Activation of the Pik1 switch occurs by a Ca2+ signal that activates the EF-hand of frequenin, which illuminates a specific PtdIns(4)P lamp. The pool of messenger lipid (different lamps) and its cellular stage (rectangular surfaces represent distinct contexts and cellular membranes onto which a light projects) determine the ‘colour’ of the signal perceived.

PtdInsStt4

Pik1 PtdIns

Ca2+

PtdIns(4)P

PtdIns(4)P

PtdIns(4,5)P2

PtdIns(3,4)P2

(4)Pkinases

© 1999 Macmillan Magazines Ltd

news and views

NATURE CELL BIOLOGY | VOL 1 | AUGUST 1999 | cellbio.nature.com E95

Durham, North Carolina 27710, USA.e-mail: yorkj@acpub.duke.edu

1. Olshevskaya, E. V., Hughes, R. E., Hurley, J. B. & Dizhoor, A. M.

J. Biol. Chem. 272, 14327–14333 (1997).

2. Pongs, O. et al. Neuron 11, 15–28 (1993).

3. Hendricks, K. B., Wang, B. Q., Schnieders, E. A. & Thorner, J.

Nature Cell Biol. 1, 234–241 (1999).

4. Zozulya, S. & Stryer, L. Proc. Natl Acad. Sci. USA 89, 11569–

11573 (1992).

5. Dizhoor, A. M. et al. Science 25, 829–832 (1993).

6. Tanaka, T. et al. Nature 376, 444–447 (1995).

7. Flaherty, K. M., Zozulya, S., Stryer, L. & McKay, D. B. Cell 75,

709–716 (1993).

8. Ames, J. B. et al. Nature 38, 198–202 (1997).

9. De Camilli, P., Emr, S. D., McPherson, P. S. & Novick, P. Science

271, 1533–1539 (1996).

10. Furman, D. A., Meyers, R. E. & Cantley, L. C. Annu. Rev.

Biochem. 67, 481–507 (1998).

11. Lee, S. B. & Rhee, S. G. Curr. Opin. Cell Biol. 7, 183–189

(1995).

12. Guo, S., Stolz, L. E., Lemrow, S. E. & York, J. D. J. Biol. Chem.

274, 12990–12995 (1999).

13. Yoshida, S., Ohya, Y., Nakano, A. & Anraku, Y. Mol. Gen. Genet.

242, 631–640 (1994).

14. Desrivieres, S. Cooke, F. T., Parker, P. J. & Hall, M. N. J. Biol.

Chem. 273, 15787–15793 (1998).

15. York, J. D. et al. Science 285, 96–100 (1999).

Vessels vivified by Akt acting on NO synthase

Solomon H. Snyder and Samie R. Jaffrey

The short- and long-term effects of stimuli such as shear stress on blood vessels are mediated through endothelial nitric oxide synthase. But whereas the short-term effects occur through transient increases in cytosolic calcium, the long-term changes result from constitutive activation of the synthase by the serine/threonine kinase Akt.

itric oxide (NO) was first identified asthe endothelial-derived relaxing factorthat is responsible for the rapid vasodi-

lation — relaxation or enlargement of bloodvessels — induced by acetylcholine, bradyki-nin and other agents. But NO also has long-term effects on cell growth and survival,including the remodelling of blood vessels,prevention of atherosclerosis and inhibitionof programmed cell death, or apoptosis.Such short- and long-term events result fromthe temporary or constitutive production,respectively, of NO. How can the cell pro-duce NO in two such different ways?

The rapid, short-term production of NO— and consequent temporary vasodilation— that occurs in response to agents such asacetylcholine is mediated through a transientincrease in the entry of calcium into cells(Fig. 1) and the binding of calcium–calmod-ulin complexes to the NO-generatingenzyme endothelial NO synthase (eNOS),which is transiently activated. But the long-term production of NO, in response to, forexample, the increased blood flow thatoccurs during exercise, is calcium independ-ent. Now two papers1,2 show that extendedNO production involves activation of phos-phatidylinositol-3-OH kinase (PI(3)K) andits effector, the serine/threonine kinase Akt,and subsequent constitutive activation ofeNOS (Fig. 1).

The principal determinant of NO pro-duction in blood vessels is shear stress3, thename given to the pressure exerted onendothelial cells by the flow of blood overthem. Acute increases in shear stress, as wellas substances such as acetylcholine, can cause

calcium influx, which probably accounts forthe early, rapid increase in NO productionthat occurs in response to such stress. How-ever, after blood flow and cytosolic calciumreturn to basal levels, NO productionbecomes constitutive, no longer dependenton receptor-stimulated calcium influx. Shearstress that is elicited by laminar — non-tur-bulent — flow strongly stimulates NO pro-duction, whereas chaotic flow, which causesfluctuating wall stresses, is a weak stimulator

N

of NO generation. Chaotic flow occurs atarterial bifurcations and curvatures, sites thatare also prone to atherosclerotic lesions. Theability of NO to decrease inflammation andinhibit myointimal proliferation, a precursorto formation of atherosclerotic plaques,means that NO is likely to be a key anti-atherogenic molecule.

How can shear stress lead to constitutiveNO production by eNOS? An earlier study byZeiher and co-workers4 showed that thePI(3)K/Akt signalling pathway, known totransduce signals from certain growth fac-tors, is also activated by shear-stress stimuli.Zeiher and co-workers have now askedwhether the PI(3)K/Akt pathway mediatesthe known effects of shear stress on eNOS2.They find that wortmannin, a potent PI(3)Kinhibitor, abolishes shear-stress-inducedproduction of NO. Shear stress normallyleads to eNOS phosphorylation, and this isblocked by wortmannin too. Zeiher andcolleagues2 also show that Akt, whose activa-tion is enhanced by the PI(3)K product phos-phatidylinositol-3,4,5-trisphosphate,mediates the effects of PI(3)K on eNOS:dominant-negative Akt molecules blockeNOS phosphorylation in response to shear

Acetylcholine, bradykinin

Ca2+

Calmodulin

Shear stress, VEGF

PI(3)K

PtdIns(3)P

PKB/Akt

eNOS

NO

cGMP, nitrosylation

Smooth-muscle relaxation

Inhibition of proliferation

Vascular remodelling

Figure 1 Production of nitric oxide (NO) is usually thought to be triggered by the increases in cytosolic calcium (Ca2+) that occur following the stimulation of receptors for molecules such as acetylcholine and bradykinin. Ca2+ forms a complex with calmodulin, and together they bind to and activate endothelial nitric oxide synthase (eNOS), which produces NO. New results1,2 indicate that important physiological triggers of NO production may actually activate eNOS through a Ca2+-independent pathway involving phosphatidylinositol-3-OH kinase (PI(3)K) and the serine/threonine kinase Akt. Activation of the PI(3)K/Akt pathway leads to eNOS phosphorylation and constitutive NO production. cGMP, cyclic GMP.