Biological s y s t e m s e x h i b i t i n g t r a n s d u c t i o n c r o s s t a l k
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6 Wegener, A-M. K. et al. (1992) Cell 68, 83-95 7 Letourneur,F. and Klausner, R. D. (1992) Science 255, 79-82 8 Baniyash, M. et al. (1988) J. Biol. Chem. 263, 18225-18230 9 June, C. H., Fletcher, M. C., Ledbetter, J. A. and Samelson, L. E. (1990) J. Immunol. 144, 1591-1599 10 Mustelin, T., Coggeshall,K, M., Isakov, N. and Altman, A. (1990) Science 247, 1584-1587 11 UIIrich,A. and Schlessinger,J. (1990) Cell 61, 203-212 12 Park, D. J., Rho, H. W. and Rhee, S. G. (1991) Proc. Natl Acad. Sci. USA 88, 5453-5456 13 Weiss, A., Koretzky, G., Schatzman,R. C. and Kadlecek, T. (1991) Proc. Natl Acad. Sci. USA 88, 5484-5488 14 Secrist, J. P., Karnitz, L. and Abraham, R. T. (1991) J. Biol. Chem. 266, 12135-12139 15 Granja, C. et al. (1991) J. Biol. Chem. 266, 16277-16280 16 Koch, C. A. et al. (1991) Science 252, 668-674 17 Samelson, L. E., Phillips,A. F., Luong, E. T. and Klausner, R. D. (1990) Proc. Natl Acad. Sci. USA 87, 4358-4362
18 Cooke, M. P., Abraham, K. M., Forbush, K. A. and Perlmutter, R. M. (1991) Cell 65, 281-291
participates in signal relay from the plasma membrane to the cytoplasm. Ongoing studies will soon answer many of the questions regarding the biochemistry of TCR signaling. A major goal for future investigations will be to understand the linkage between specific signaling events and the various effector functions of activated T cells.
References 1 Finkel,T. H., Kubo, R. T. and CambierJ. C. (1991) Immunol. Today 12, 79-85 2 Manolios, N., Letourneur, F., Bonifacino,J. S. and Klausner, R. D. (1991) EMBO J. 10, 1643-1651 3 Keegan,A. D. and Paul, W. E. (1992) Immunol. Today 13, 63-68 4 Irving, B. A. and Weiss, A. (1991) Cell 64, 891-901 5 Romeo, C., Amiot, M. and Seed, B. (1992) Cell 68, 889-897
THE ACTIN CYTOSKELETON is composed of a complicated meshwork of microfilaments whose precise geometry is regulated by a large cadre of actinbinding proteins. These proteins are responsible for many characteristic structures including stress fibers, lamellipodia, filopodia and the cortical actin network. Regulated changes in the actin cytoskeleton occur in a large number of biological responses including mitogenesis, morphogenesis, movement and secretion; however, the signal transduction pathways leading to actin remodeling are poorly understood. Our lack of information is mainly due to the complexity of the events. For example, when a cell migrates, a host of signals must be processed, which ultimately result in motion. Chemotactic receptors provide the vectorial coordinates for directed motion. Actin filament turnover is associated with the formation of microspikes and lamellipodia at the leading edge. Contact between the actin cytoskeleton and adhesion receptors, and between the adhesion receptors and the extracellular matrix must be modulated both to provide traction for forward movement at the leading edge of the cell, and anchorage to which its trailing regions are drawn. In addition, changes in actin structure must be coordinated with changes in microA. Aderem is at the Laboratory of Signal Transduction, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.
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19 Davidson, D., Chow, L. M. L., Fournel, M. and Veillette, A. (1992) J. Exp. Med. 175, 1483-1492 20 Sefton, B. M. (1990) Oncogene 6, 683-686 21 Koretzky,G. A., Picus, J., Thomas, M. L. and Weiss, A. (1990) Nature 346, 66-68 22 Caron, L., Abraham, N., Pawson,T. and Veillette, A. (1992) Mol. Cell. Biol. 12, 2720-2729 23 Karnitz, L. et al. Mol. Cell. Biol. (in press) 24 Kypta, R. M., Goldberg,Y., Ulug, E. T. and Courtneidge,S. A. (1990) Cell 62,481-492 25 Chan, A. C., Irving, B. A., Fraser, J. D. and Weiss, A. (1991) Proc. Natl Acad. Sci. USA 88, 9166-9170 26 Wange, R. L., Kong,A-N.T. and Samelson, L. E. (1992) J. Biol. Chem. 267, 11685-11688 27 Hutchcroft, J. E., Harrison, M. L. and Geahlen, R. L. (1991) J. Biol. Chem. 266, 14846-14849 28 Ettehadieh, E. et al. (1992) Science 255, 853-855 29 Downward,J. et al. (1990) Nature 346, 719-723
Signal transduction and the actin cytoskeleton: the roles of MARCKS and profilin
MARCKS and profilin, two actin-binding proteins, are d i s c u s s e d to illustrate the m e c h a n i s m by which extracellular signals are coupled to changes in the structure of the actin cytoskeleton, MARCKS is a f i l a m e n t o u s actincrosslinking protein t h a t appears to function as an integrator of protein kinase C and calcium (Ca2+)/calmodulin signals in the regulation of a c t i n m e m b r a n e interactions. New data suggest t h a t profilin is activated by the coordinated action of receptor tyrosine kinases and p h o s p h o l i p a s e C-71 to s t i m u l a t e the stabilization of actin filaments.
(villin), (3) block the barbed end of the filament (fragmin), (4) block the pointed end of the filament (]3-actinin), (5) sever the filament (geisolin) and (6) 'nibble' at the filament (depactin) 1. Second, Factin can be crosslinked to form strucActin-bindingproteins regulate actin tures of higher complexity, including structure The physical state of actin in the cell bundles and networks 2. These strucis regulated by three reversible cycles 1-3. tures give tensile strength to proFirst, monomeric ((3-) actin polymerizes trusions such as filopodia and microto form filamentous (F-) actin and F- villi and contribute to the structure of actin depolymerizes to generate actin the cell surface. This cycle is regulated monomersL This cycle is regulated by by at least four classes of actin crosssix classes of proteins which (1) se- linking proteins that organize actin filaquester actin monomers (e.g. profilin), ments into tight bundles (villin), organ(2) control nucleation of the filament ize actin filaments into loose bundles © 1992,ElsevierSciencePublishers,(UK) 0376-5067/92/$05.00 tubules and intermediate filaments; organelles must be reoriented and energy stores have to be generated in the appropriate cellular domains.
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Biological systems exhibiting t r a n s d u c t i o n
((z-actinin), organize actin filaments into orthogonal networks [actin-binding protein (ABP)] and cross-link actin oligomers (spectrin) 1,2. Third, actin structures are reversibly anchored to the membrane. This cycle is facilitated by a class of actin-binding proteins, which include MARCKS (myristoylated, alanine-rich C kinase substrate) and ABP, whose affinity for the membrane is regulated. The function of the almost 70 actinbinding proteins described are regulated by second messengers, kinases, bioactive lipids and phospholipids 1-3. Calcium (Ca2+), for example, regulates the crosslinking of actin filaments by nonmuscle c~-actinins and activates the nucleating, severing and filament endblocking activities of villin, severin and gelsolin, respectively< Similarly, phosphorylation by cyclic AMP (cAMP)dependent protein kinase reversibly inhibits the bundling of actin filaments by synapsin I and by erythrocyte band 4.9 (Ref. 1). A discussion on the crosstalk that occurs between the plasma membrane and the myriad of actinbinding proteins is beyond the scope of this brief review. Instead, I will focus on new data dealing with two actin-binding proteins, MARCKS and profilin, whose attachment to the membrane and whose actin-binding capacities are regulated by different signal transduction pathways.
MARCKS Phosphorylation-dependent cycling. MARCKS is a specific protein kinase C (PKC) substrate which is targeted to the membrane by its amino-terminal, myristoylated membrane-binding domain4. This positions the substrate dose to PKC5, facilitating its efficient phosphorylation. PKC-dependent phosphorylation displaces MARCKS from the membrane and its subsequent dephosphorylation is accompanied by its reassociation with the membrane6,7. MARCKS has a punctate distribution in macrophages, and many of the structures containing MARCKS are found at the substrate-adherent surface of pseudopodia and filopodia 5. Many of these structures also contain vinculin and talin, known components of focal contacts. Immunoelectron microscopy shows MARCKS to be in clusters at points where actin filaments interact with the cytoplasmic surface of the plasma membrane (A. Rosen, J. Hartwig, A. C. Nairn and A. Aderem, submitted). Phosphorylation of MARCKS results in its translocation from membrane-bound structures to the cytosol, where it remains closely associated with actin filaments. The capacity of MARCKS to shuttle reversibly between membrane and cytosol, together with the observation that MARCKSis localized at the membrane in discrete structures 5;, sug-
crosstalk
gests that the protein associates with the membrane through a receptor, rather than through non-specific insertion of the myristic acid in the lipid bilayer. Calmodulin-binding and actin filament crosslinking. MARCKS binds both calmodulin and actin in a complex manner8'9. MARCKS will only bind to calmodulin in the presence of calcium and the phosphorylation of MARCKS prevents the binding of Ca2+/calmodulin to it< MARCKSalso binds to the sides of actin filaments and crosslinks them, and this activity is inhibited by phosphorylation or by Ca2+/caimodulin9. Filament crosslinking requires MARCKSto dimerize or have two actin-binding sites Accordingly, phosphorylation or Ca2+/calmodulin presumably inactivates one of the actinbinding sites or dissociates the dimers. The competing interaction of calmodulin, PKC and actin can be explained by the structure of the effector domain of MARCKS,located in the middle of the rod-shaped molecule (Fig. 1). This highly conserved domain can be modeled as an amphipathic (z-helix, and the five Lys residues positioned on one side of the helix constitute both the calmodulin and actin-binding sites of MARCKS, hence the competition between these two ligands 8,9. The three Ser residues positioned at the opposite side of the helix are substrates for PKC, and their
Phosphorylation C a l m o d u l i n binding Actin b i n d i n g
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Figure 1 The domain structure of MARCKS. MARCKS is a rod-shaped molecule with two highly conserved domains. The myristoylated amino-terminal membrane-binding domain and the effector domain, which bears the phosphorylation sites and the calmodulin- and actin-binding sites are indicated. The effector domain is also represented as a helical wheel showing the Phe residues that contribute to its amphipathic structure (_F),the Lys residues that form the calmodulin- and actin-binding sites (+) and the Ser residues phosphorylated by PKC (*). The numbering of the amino acids in the helical wheel is for murine MARCKS. The peptide depicted by the helical wheel binds calmodulin and actin in vitro and this binding is regulated by PKC-dependent phosphorylation.
439
Biologicalsystemsexhibitingtransductioncrosstalk
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OCTOBER1992
Calciun
proteins Ph°sph~/
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crosslinked actin at the membrane
-Pi
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uncrosslinked actin in the cytosol
Figure 2 Model indicating a possible mechanism by which MARCKSmight regulate actin-membrane interaction. Details of the model are described in the text, Agonist receptor activates PKC through a cascade involving G proteins (G) and phospholipase C (PLC). PKC phosphorylates MARCKS (M) which is released from its membrane receptor (R). An increase in cellular Ca 2+ results in the binding of calmodulin (Cal) to MARCKS.
phosphorylation prevents the binding of calmodulin or actin, possibly because the negative phosphate groups influence the positively charged Lys residues (Fig. 1). The proximity of the sites also explains why Ca2÷/calmodulin prevents the phosphorylation of MARCKS8. Integration of Ca2÷ and PKC signals. Figure 2 presents a model for MARCKS function which is consistent with current data. At rest, MARCKS associates with the cytoplasmic face of the membrane, probably by binding to a receptor molecule 7. In its non-phosphorylated form MARCKS crosslinks actin, so it is likely that the actin meshwork associated with the membrane via MARCKS is relatively rigid 9. Activated PKC phosphorylates MARCKS, which is released from the membrane 6,7. Phosphorylated, cytosolic MARCKS still associates with actin filaments, but can no longer crosslink actin 9. The actin linked to MARCKS is likely to be spatially separated from the membrane and more plastic. When MARCKS is dephosphorylated, it returns to the membrane, where it once again crosslinks actin 7,9. An increase in
440
that promotes the gelation of actin filai n t r a c e l l u l a r Ca 2÷ would promote the binding of calmodulin to MARCKS8, ments into orthogonal networks under which would inhibit its actin crosslinking the cell surface, binds to immunoglobulin activity9. This would result in less rigid G (IgG) Fc receptor I (FcTRI)11. Ligation actin, still linked through MARCKS to of the receptor with IgG results in a the membrane. A decrease in intracellu- decreased avidity of FcTRI for ABP. lar Ca 2+ would shift the equilibrium back Thus ABP molecules could function to to the resting state, where MARCKS once both crosslink actin filaments on the again could crosslink actin at the mem- cytoplasmic face of the membrane and brane. Since Ca 2+ levels are known to to link the cross]inked actin gel to the oscillate following cellular stimulation 1°, bilayer by association with FcTRI. LigMARCKS would mediate cycles of actin ation of the receptor would result in the crosslinking activity at the membrane. release of ABP from the membrane and However, when MARCKS is phosphory- in the separation of the actin lattice lated it is unable to bind calmodulin 8 from the membrane. and is released from the membrane 7. It should also be noted that second Thus, PKC would induce a local destabil- messengers such as diacylglycerol (DAG) ization of the actin skeleton through the a n d Ca 2+ can regulate actin structure through a number of additional routes. phosphorylation of MARCKS. This hypothetical model should not be For example, DAG, in conjunction with interpreted as indicating that MARCKS an as yet undefined protein, has recently alone regulates both the reversible been shown to stimulate the nucleation attachment of actin to the membrane of actin filaments at the membrane 12. and the physical structure of the actin Ca2+, as mentioned above, regulates the network. This task is clearly shared by activity of a great many additional a large number of other actin-binding actin-binding proteins. Function of MARCKS. The context in proteins. For example, it has recently been shown that ABP, a homodimer which MARCKS might regulate actin-
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Biological systems exhibiting transduction
chemotaxis, and it is significant that membrane interactions is suggested by the chemotactic agents f-Met-Leu-Phe the agonists that promote its phosand interleukin 8 (IL-8) cause MARCKS phorylation and the conditions under to shuttle between the membrane which its synthesis is induced. MARCKS and the cytosol in neutrophils 7. is phosphorylated during chemotaxis, secretion and phagocytosis in neutro- (2) Secretion and membrane traffic. The phils and macrophages ~3,~4,during neurorole of MARCKS does not appear to secretion o,t5 and during mitogenesis t6'~7. be restricted to the regulation of actin in motile structures. It is found Each of these events is accompanied by in high concentration in presynaptic a rearrangement of the actin cytoskeljunctions and is phosphorylated eton. The role of MARCKS in these variwhen synaptosomes are depolarous systems will be briefly evaluated. ized6,15, suggesting a function in secretion or membrane recycling. In (1) Motility. MARCKS co-localizes with addition, both tumor necrosis factor vinculin, talin and PKC at the suband bacterial lipopolysaccharide strate-adherent surface of macroinduce the synthesis of MARCKS phage filopodia and activation of and concomitantly prime macroPKC results in the displacement of MARCKS from these focal contacts phages and neutrophils for enhanced PKC-dependent responses and in the rearrangement of the such as the secretion of inflammatory actin cytoskeletons. MARCKS might, mediators and cytokines t3,'4. therefore, provide a PKC-sensitive reversible crossbridge between the (3) Mitogenesis and transformation. A actin cytoskeleton and the plasma number of neuropeptides that act as growth factors in Swiss 3T3 cells membrane. Such a reversible association of actin is a prerequisite for also induce the phosphorylation of MARCKS18, although it has yet to be directed cellular locomotion during
crosstalk
proven that this event is an intermediate in the mitogenic pathway. A number of reports have suggested that the level of MARCKS is down regulated by transformation 19. A change in cell shape is one of the hallmarks of transformation: cells become much less adherent and round up, there is a marked reduction in the number and thickness of actin stress fibers and the cells become capable of anchorageindependent growth. MARCKS may therefore have a role in maintaining normal cell shape or in delivering anchorage-dependent growth signals, although it is equally possible that its down-regulation during transformation is a consequence of shape change. Profilin Promotion of F-actin stabilization. Profilin is a 12-15 kDa protein that binds monomeric actin and, at high profilin to actin ratios, prevents actin filament formation by monomer sequestration 3,2°,21.
'l'Ca 2+ Cytoskeletal remodeling
Other ADP
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actin-binding proteins
G-actin Stable actin filaments
Figure 3 Model of how RTKs may regulate the activity of profilin. In unstimulated cells profilin (Prof) binds tightly to phosphatidylinositol 4,5-bisphosphate [Ptdlns(4,5)P2]. This prevents its participation in cytoskeletal remodeling and blocks access of unpflosphorylated phospholipase C5'$ (PLC-71) to Ptdlns(4,5)P 2. Upon addition of EGF, its receptor dimerizes and the activated protein tyrosine kinase (PTK) phosphoryiates itself and tyrosine residues on PLC-~'I. Phosphorylated phospholipase C-~'1(PLC-y-P) can hydrolyse profilin-bound Ptdlns(4,5)P 2 to produce diacylglycerol (DAG), which activates PKC, and inositol trisphosphate (InsP3), which triggers an increase in intracellular Ca2+. The hydrolysis of Ptdlns(4,5)P 2 results in the release of profilin from the membrane allowing it to catalyse adenine nucleotide exchange on G-actin, which promotes stable actin filaments. 441
Biological systems exhibiting transduction crosstalk However, both the cellular levels of profilin, as well as its affinity for actin, appear too low for it to play this role in vivo2°." The role of profilin in vivo is more likely to be related to its capacity to catalyse the exchange of adenine nucleotides on monomeric actin 2°. During actin filament formation, ATP bound to G-actin is hydrolysed to ADP, which remains tightly bound to F-actin22. During filament remodeling, ADP-actin monomers are released. However, while ADP-actin monomers can polymerize, ATP-actin monomers do so more rapidly and form more stable filaments than their dinucleotide-bound counterparts 3,23. Since the exchange of ADP for ATP on G-actin is rate limiting, profilin has the potential to promote actin polymerization and filament stabilization by catalysing actin nucleotide exchange2°. This has been shown in cells that stably overexpress profilin, where actin filament stability, measured by the fluorescence decay of microinjected caged resorufin-actin, is directly proportional to profilin concentration (T. Finkel et al., submitted). However, the observation that microinjected profilin decreases cellular F-actin and prevents the extension of lamellipodia indicates that at high concentrations, profilin can sequester actin monomers in ViVO24. In contrast, the microinjection of highaffinity profilin-actin complexes increase F-actin and enhances membrane ruffling24, consistent with the effect of profilin on actin nucleotide exchange. Profilin binds to polyphosphoinositides.P r o filin binds tightly and specifically to four or five molecules of phosphatidylinositol 4,5-bisphosphate [Ptdlns(4,5)P2] in vitro, and this results in a dissociation of the profilin-actin complex25. Concomitantly, profilin masks PtdIns(4,5)P2 from the hydrolytic action of phospholipase C-7~(PLC-7~)26. Thus, profilin provides a link between a major signal transduction pathway, the phosphatidylinosito] (PtdIns) cycle, and the actin cytoskeleton (Fig. 3). Ultrastructural localization studies demonstrate a significant fraction of profilin in close association with both the internal leaflet of the plasma membrane and external surfaces of intracellular membranes in human leukocytes and platelets 27. This association with the membrane is likely to reflect its direct binding to phosphatidylinositol 4-phosphate [PtdIns4P] and PtdIns(4,5)P2. Stimulation of platelets with thrombin results in rapid PtdIns turnover, the cycling of profilin between the cytosol 442
and the plasma membrane, changes in the levels of the high-affinity profilactin complex and in the polymerization of actin 27. However, it is important to recognize that a number of other actinbinding proteins, including gelsolin, gCap39, severin and protein 4.1 also bind to PtdIns(4,5)P2 (Ref. 28), and any scheme devised to explain the connection of the PtdIns cycle to actin remodeling would also have to account for the actions of these other actinbinding proteins. The receptor tyrosine kinase-profilin connection. As discussed above, PtdIns(4,S)P2 that is bound to profilin is not hydrolysed by PLC-7~26 (Fig. 3). However, when PLC-Tx is phosphorylated by the action of receptor tyrosine kinases (RTKs), such as the receptors for epidermal growth factor (EGF) or platelet derived growth factor (PDGF), it becomes capable of hydrolysing profilinbound Ptdlns(4,5)P2 to generate inosito] trisphosphate [Ins(1,4,5)P3] and diacylglycerol29. This suggests that profilin is now free to interact with actin and stabilize filaments (Fig. 3). It is well known that growth factors such as EGF and PDGF influence the actin cytoskeleton and induce cells to change shape, ruffle their membranes, migrate and divide. However, the mechanism by which RTKs influence the organization of the actin cytoskeleton is unknown. Also obscure is the mechanism by which receptor tyrosine kinases activate inositol 1-phosphate (InslP) turnover, since the phosphorylation of PLC-7~ does not appreciably influence its activity in vitro 2°. Actin-binding proteins such as profilin, which bind to Ptdlns(4,5)P2 and mask it from the action of non-phosphorylated PLC-y1, might be a key intermediate in both these events. It also seems likely that other actin-binding proteins, which bind PtdIns(4,5)P2, such as gelsolin and pCap39, might similarly transduce signals from RTKs to the actin cytoskeleton. An alternative mechanism by which RTKs may influence cytoskeletal events is by direct action of the receptors on actin-binding proteins which bear Src homology 2 (SH2) domains. For example, tensin, an actin-binding protein which is a component of focal contacts, bears an SH2 domain that binds to a number of phosphotyrosine-containing proteins 3°. Tensin may therefore bind directly to autophosphorylated RTKs and mediate their direct association with the actin cytoskeleton. In addition, many RTKs activate phospholipases that generate diacylglycerol and InsP3; these two sec-
TIBS 17 - OCTOBER 1992 ond messengers, in turn, activate PKC and increase intracellular Ca2~. As discussed above, diacylglycerol can nucleate actin filaments 12, Ca 2÷ c a n regulate a large number of actin-binding proteins 1, and PKC can alter actin structure through MARCKS9. The Ras-CAP signal transduction pathway. The Ras family of low molecular weight guanine nucleotide-binding proteins has a role in signal transduction pathways that regulate growth and differentiation31 (also see other reviews in this issue). In the yeast Saccharomyces cerevisiae, Ras activates adenylate cyclase activity in a complex that consists of at least two components: the adenylate cyclase catalytic subunit and an adenylate cyclaseassociated protein (CAP)2°,32. CAP is a bifunctional signal transducer: its aminoterminal domain is necessary and sufficient for cellular responsiveness to activated Ras, and its carboxy-terminal domain has a role in a number of cellular functions including the control of cell morphology. For example, yeast ceils which are null for CAP bud randomly and are defective in actin distribution32o The morphological and nutritional defects associated with the loss of the CAP carboxy-terminal domain are suppressed by overexpression of the profilin gene 32. In addition, cells lacking profilin are abnormally enlarged and round, do not contain actin cables and bud randomly from the cell surface 21, a phenotype similar to that of cells lacking the carboxyterminal domain of CAP32. Mutational analysis and studies using Acanthamoeba profilins indicate that the ability of profilin to suppress the cap- phenotype is dependent on the ability to bind PtdIns(4,5)P2 in conjunction with its actin-binding capacity32. Thus, it appears that CAP and profilin are components of the same signaling pathway, which implies that CAP may be a component of a polyphosphoinositide signal transduction pathway. Alternatively, CAP itself may bind actin, or CAP and profilin may bind to each other. It seems likely that CAP itself is an actinbinding protein, since it is highly homologous to ASP-56, an actin monomer sequestering protein from porcine platelets 33. However, the possibility that CAP may be regulated by phospholipids is of special interest since the activity of Ras has also been shown to be regulated by bioactive phospholipids 34.
Conclusions and perspectives Advances in our understanding of signal transduction and cytoskeletal
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Biological systems exhibiting transduction
structure have allowed a glimpse of the complicated network of events which translates information from the extracellular environment into changes in cellular architecture. Rapid progress is being made in understanding the mechanisms by which individual actin-binding proteins regulat_e actin structure, and are themselves regulated. Much more daunting is the task of understanding how the more than 70 actinbinding proteins interact in vivo. Fortunately, genetic studies of yeast and flies, used in conjunction with the array of techniques already available, are facilitating progress. For example, a recent study demonstrating that the chickadee gene product, required for intercellular cytoplasm transport during Drosophila oogenesis, is homologous to profilin3s, has provided new insight into the regulation and function of actin in vivo.
supported by the NIH, the American Heart Association, and the Cancer Research Institute.
Acknowledgements
I wish to thank P. Goldschmidt for providing me with preprints of unpublished work, and J. Hartwig, P. Janmey, P. Goldschmidt, K. Barker, P. Matsudaira and A. Nairn for discussion and comments on the manuscript. I would also like to thank all the members of my laboratory for helpful discussions and for commenting on the manuscript. Research in the author's laboratory was
References Due to TIBS editorial policy, the number of references cited have been limited. I apologize to those authors whose work has been cited through the reviews of others.
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This article is dedicated to the memory of
~Ef Racker.
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6 Wegener, A-M. K. et al. (1992) Cell 68, 83-95 7 Letourneur,F. and Klausner, R. D. (1992) Science 255, 79-82 8 Baniyash, M. et al. (1988) J. Biol. Chem. 263, 18225-18230 9 June, C. H., Fletcher, M. C., Ledbetter, J. A. and Samelson, L. E. (1990) J. Immunol. 144, 1591-1599 10 Mustelin, T., Coggeshall,K, M., Isakov, N. and Altman, A. (1990) Science 247, 1584-1587 11 UIIrich,A. and Schlessinger,J. (1990) Cell 61, 203-212 12 Park, D. J., Rho, H. W. and Rhee, S. G. (1991) Proc. Natl Acad. Sci. USA 88, 5453-5456 13 Weiss, A., Koretzky, G., Schatzman,R. C. and Kadlecek, T. (1991) Proc. Natl Acad. Sci. USA 88, 5484-5488 14 Secrist, J. P., Karnitz, L. and Abraham, R. T. (1991) J. Biol. Chem. 266, 12135-12139 15 Granja, C. et al. (1991) J. Biol. Chem. 266, 16277-16280 16 Koch, C. A. et al. (1991) Science 252, 668-674 17 Samelson, L. E., Phillips,A. F., Luong, E. T. and Klausner, R. D. (1990) Proc. Natl Acad. Sci. USA 87, 4358-4362
18 Cooke, M. P., Abraham, K. M., Forbush, K. A. and Perlmutter, R. M. (1991) Cell 65, 281-291
participates in signal relay from the plasma membrane to the cytoplasm. Ongoing studies will soon answer many of the questions regarding the biochemistry of TCR signaling. A major goal for future investigations will be to understand the linkage between specific signaling events and the various effector functions of activated T cells.
References 1 Finkel,T. H., Kubo, R. T. and CambierJ. C. (1991) Immunol. Today 12, 79-85 2 Manolios, N., Letourneur, F., Bonifacino,J. S. and Klausner, R. D. (1991) EMBO J. 10, 1643-1651 3 Keegan,A. D. and Paul, W. E. (1992) Immunol. Today 13, 63-68 4 Irving, B. A. and Weiss, A. (1991) Cell 64, 891-901 5 Romeo, C., Amiot, M. and Seed, B. (1992) Cell 68, 889-897
THE ACTIN CYTOSKELETON is composed of a complicated meshwork of microfilaments whose precise geometry is regulated by a large cadre of actinbinding proteins. These proteins are responsible for many characteristic structures including stress fibers, lamellipodia, filopodia and the cortical actin network. Regulated changes in the actin cytoskeleton occur in a large number of biological responses including mitogenesis, morphogenesis, movement and secretion; however, the signal transduction pathways leading to actin remodeling are poorly understood. Our lack of information is mainly due to the complexity of the events. For example, when a cell migrates, a host of signals must be processed, which ultimately result in motion. Chemotactic receptors provide the vectorial coordinates for directed motion. Actin filament turnover is associated with the formation of microspikes and lamellipodia at the leading edge. Contact between the actin cytoskeleton and adhesion receptors, and between the adhesion receptors and the extracellular matrix must be modulated both to provide traction for forward movement at the leading edge of the cell, and anchorage to which its trailing regions are drawn. In addition, changes in actin structure must be coordinated with changes in microA. Aderem is at the Laboratory of Signal Transduction, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.
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19 Davidson, D., Chow, L. M. L., Fournel, M. and Veillette, A. (1992) J. Exp. Med. 175, 1483-1492 20 Sefton, B. M. (1990) Oncogene 6, 683-686 21 Koretzky,G. A., Picus, J., Thomas, M. L. and Weiss, A. (1990) Nature 346, 66-68 22 Caron, L., Abraham, N., Pawson,T. and Veillette, A. (1992) Mol. Cell. Biol. 12, 2720-2729 23 Karnitz, L. et al. Mol. Cell. Biol. (in press) 24 Kypta, R. M., Goldberg,Y., Ulug, E. T. and Courtneidge,S. A. (1990) Cell 62,481-492 25 Chan, A. C., Irving, B. A., Fraser, J. D. and Weiss, A. (1991) Proc. Natl Acad. Sci. USA 88, 9166-9170 26 Wange, R. L., Kong,A-N.T. and Samelson, L. E. (1992) J. Biol. Chem. 267, 11685-11688 27 Hutchcroft, J. E., Harrison, M. L. and Geahlen, R. L. (1991) J. Biol. Chem. 266, 14846-14849 28 Ettehadieh, E. et al. (1992) Science 255, 853-855 29 Downward,J. et al. (1990) Nature 346, 719-723
Signal transduction and the actin cytoskeleton: the roles of MARCKS and profilin
MARCKS and profilin, two actin-binding proteins, are d i s c u s s e d to illustrate the m e c h a n i s m by which extracellular signals are coupled to changes in the structure of the actin cytoskeleton, MARCKS is a f i l a m e n t o u s actincrosslinking protein t h a t appears to function as an integrator of protein kinase C and calcium (Ca2+)/calmodulin signals in the regulation of a c t i n m e m b r a n e interactions. New data suggest t h a t profilin is activated by the coordinated action of receptor tyrosine kinases and p h o s p h o l i p a s e C-71 to s t i m u l a t e the stabilization of actin filaments.
(villin), (3) block the barbed end of the filament (fragmin), (4) block the pointed end of the filament (]3-actinin), (5) sever the filament (geisolin) and (6) 'nibble' at the filament (depactin) 1. Second, Factin can be crosslinked to form strucActin-bindingproteins regulate actin tures of higher complexity, including structure The physical state of actin in the cell bundles and networks 2. These strucis regulated by three reversible cycles 1-3. tures give tensile strength to proFirst, monomeric ((3-) actin polymerizes trusions such as filopodia and microto form filamentous (F-) actin and F- villi and contribute to the structure of actin depolymerizes to generate actin the cell surface. This cycle is regulated monomersL This cycle is regulated by by at least four classes of actin crosssix classes of proteins which (1) se- linking proteins that organize actin filaquester actin monomers (e.g. profilin), ments into tight bundles (villin), organ(2) control nucleation of the filament ize actin filaments into loose bundles © 1992,ElsevierSciencePublishers,(UK) 0376-5067/92/$05.00 tubules and intermediate filaments; organelles must be reoriented and energy stores have to be generated in the appropriate cellular domains.
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Biological systems exhibiting t r a n s d u c t i o n
((z-actinin), organize actin filaments into orthogonal networks [actin-binding protein (ABP)] and cross-link actin oligomers (spectrin) 1,2. Third, actin structures are reversibly anchored to the membrane. This cycle is facilitated by a class of actin-binding proteins, which include MARCKS (myristoylated, alanine-rich C kinase substrate) and ABP, whose affinity for the membrane is regulated. The function of the almost 70 actinbinding proteins described are regulated by second messengers, kinases, bioactive lipids and phospholipids 1-3. Calcium (Ca2+), for example, regulates the crosslinking of actin filaments by nonmuscle c~-actinins and activates the nucleating, severing and filament endblocking activities of villin, severin and gelsolin, respectively< Similarly, phosphorylation by cyclic AMP (cAMP)dependent protein kinase reversibly inhibits the bundling of actin filaments by synapsin I and by erythrocyte band 4.9 (Ref. 1). A discussion on the crosstalk that occurs between the plasma membrane and the myriad of actinbinding proteins is beyond the scope of this brief review. Instead, I will focus on new data dealing with two actin-binding proteins, MARCKS and profilin, whose attachment to the membrane and whose actin-binding capacities are regulated by different signal transduction pathways.
MARCKS Phosphorylation-dependent cycling. MARCKS is a specific protein kinase C (PKC) substrate which is targeted to the membrane by its amino-terminal, myristoylated membrane-binding domain4. This positions the substrate dose to PKC5, facilitating its efficient phosphorylation. PKC-dependent phosphorylation displaces MARCKS from the membrane and its subsequent dephosphorylation is accompanied by its reassociation with the membrane6,7. MARCKS has a punctate distribution in macrophages, and many of the structures containing MARCKS are found at the substrate-adherent surface of pseudopodia and filopodia 5. Many of these structures also contain vinculin and talin, known components of focal contacts. Immunoelectron microscopy shows MARCKS to be in clusters at points where actin filaments interact with the cytoplasmic surface of the plasma membrane (A. Rosen, J. Hartwig, A. C. Nairn and A. Aderem, submitted). Phosphorylation of MARCKS results in its translocation from membrane-bound structures to the cytosol, where it remains closely associated with actin filaments. The capacity of MARCKS to shuttle reversibly between membrane and cytosol, together with the observation that MARCKSis localized at the membrane in discrete structures 5;, sug-
crosstalk
gests that the protein associates with the membrane through a receptor, rather than through non-specific insertion of the myristic acid in the lipid bilayer. Calmodulin-binding and actin filament crosslinking. MARCKS binds both calmodulin and actin in a complex manner8'9. MARCKS will only bind to calmodulin in the presence of calcium and the phosphorylation of MARCKS prevents the binding of Ca2+/calmodulin to it< MARCKSalso binds to the sides of actin filaments and crosslinks them, and this activity is inhibited by phosphorylation or by Ca2+/caimodulin9. Filament crosslinking requires MARCKSto dimerize or have two actin-binding sites Accordingly, phosphorylation or Ca2+/calmodulin presumably inactivates one of the actinbinding sites or dissociates the dimers. The competing interaction of calmodulin, PKC and actin can be explained by the structure of the effector domain of MARCKS,located in the middle of the rod-shaped molecule (Fig. 1). This highly conserved domain can be modeled as an amphipathic (z-helix, and the five Lys residues positioned on one side of the helix constitute both the calmodulin and actin-binding sites of MARCKS, hence the competition between these two ligands 8,9. The three Ser residues positioned at the opposite side of the helix are substrates for PKC, and their
Phosphorylation C a l m o d u l i n binding Actin b i n d i n g
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Figure 1 The domain structure of MARCKS. MARCKS is a rod-shaped molecule with two highly conserved domains. The myristoylated amino-terminal membrane-binding domain and the effector domain, which bears the phosphorylation sites and the calmodulin- and actin-binding sites are indicated. The effector domain is also represented as a helical wheel showing the Phe residues that contribute to its amphipathic structure (_F),the Lys residues that form the calmodulin- and actin-binding sites (+) and the Ser residues phosphorylated by PKC (*). The numbering of the amino acids in the helical wheel is for murine MARCKS. The peptide depicted by the helical wheel binds calmodulin and actin in vitro and this binding is regulated by PKC-dependent phosphorylation.
439
Biologicalsystemsexhibitingtransductioncrosstalk
TIBS17-
OCTOBER1992
Calciun
proteins Ph°sph~/
Key mem
~
crosslinked actin at the membrane
-Pi
~
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uncrosslinked actin at the membrane
uncrosslinked actin in the cytosol
Figure 2 Model indicating a possible mechanism by which MARCKSmight regulate actin-membrane interaction. Details of the model are described in the text, Agonist receptor activates PKC through a cascade involving G proteins (G) and phospholipase C (PLC). PKC phosphorylates MARCKS (M) which is released from its membrane receptor (R). An increase in cellular Ca 2+ results in the binding of calmodulin (Cal) to MARCKS.
phosphorylation prevents the binding of calmodulin or actin, possibly because the negative phosphate groups influence the positively charged Lys residues (Fig. 1). The proximity of the sites also explains why Ca2÷/calmodulin prevents the phosphorylation of MARCKS8. Integration of Ca2÷ and PKC signals. Figure 2 presents a model for MARCKS function which is consistent with current data. At rest, MARCKS associates with the cytoplasmic face of the membrane, probably by binding to a receptor molecule 7. In its non-phosphorylated form MARCKS crosslinks actin, so it is likely that the actin meshwork associated with the membrane via MARCKS is relatively rigid 9. Activated PKC phosphorylates MARCKS, which is released from the membrane 6,7. Phosphorylated, cytosolic MARCKS still associates with actin filaments, but can no longer crosslink actin 9. The actin linked to MARCKS is likely to be spatially separated from the membrane and more plastic. When MARCKS is dephosphorylated, it returns to the membrane, where it once again crosslinks actin 7,9. An increase in
440
that promotes the gelation of actin filai n t r a c e l l u l a r Ca 2÷ would promote the binding of calmodulin to MARCKS8, ments into orthogonal networks under which would inhibit its actin crosslinking the cell surface, binds to immunoglobulin activity9. This would result in less rigid G (IgG) Fc receptor I (FcTRI)11. Ligation actin, still linked through MARCKS to of the receptor with IgG results in a the membrane. A decrease in intracellu- decreased avidity of FcTRI for ABP. lar Ca 2+ would shift the equilibrium back Thus ABP molecules could function to to the resting state, where MARCKS once both crosslink actin filaments on the again could crosslink actin at the mem- cytoplasmic face of the membrane and brane. Since Ca 2+ levels are known to to link the cross]inked actin gel to the oscillate following cellular stimulation 1°, bilayer by association with FcTRI. LigMARCKS would mediate cycles of actin ation of the receptor would result in the crosslinking activity at the membrane. release of ABP from the membrane and However, when MARCKS is phosphory- in the separation of the actin lattice lated it is unable to bind calmodulin 8 from the membrane. and is released from the membrane 7. It should also be noted that second Thus, PKC would induce a local destabil- messengers such as diacylglycerol (DAG) ization of the actin skeleton through the a n d Ca 2+ can regulate actin structure through a number of additional routes. phosphorylation of MARCKS. This hypothetical model should not be For example, DAG, in conjunction with interpreted as indicating that MARCKS an as yet undefined protein, has recently alone regulates both the reversible been shown to stimulate the nucleation attachment of actin to the membrane of actin filaments at the membrane 12. and the physical structure of the actin Ca2+, as mentioned above, regulates the network. This task is clearly shared by activity of a great many additional a large number of other actin-binding actin-binding proteins. Function of MARCKS. The context in proteins. For example, it has recently been shown that ABP, a homodimer which MARCKS might regulate actin-
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Biological systems exhibiting transduction
chemotaxis, and it is significant that membrane interactions is suggested by the chemotactic agents f-Met-Leu-Phe the agonists that promote its phosand interleukin 8 (IL-8) cause MARCKS phorylation and the conditions under to shuttle between the membrane which its synthesis is induced. MARCKS and the cytosol in neutrophils 7. is phosphorylated during chemotaxis, secretion and phagocytosis in neutro- (2) Secretion and membrane traffic. The phils and macrophages ~3,~4,during neurorole of MARCKS does not appear to secretion o,t5 and during mitogenesis t6'~7. be restricted to the regulation of actin in motile structures. It is found Each of these events is accompanied by in high concentration in presynaptic a rearrangement of the actin cytoskeljunctions and is phosphorylated eton. The role of MARCKS in these variwhen synaptosomes are depolarous systems will be briefly evaluated. ized6,15, suggesting a function in secretion or membrane recycling. In (1) Motility. MARCKS co-localizes with addition, both tumor necrosis factor vinculin, talin and PKC at the suband bacterial lipopolysaccharide strate-adherent surface of macroinduce the synthesis of MARCKS phage filopodia and activation of and concomitantly prime macroPKC results in the displacement of MARCKS from these focal contacts phages and neutrophils for enhanced PKC-dependent responses and in the rearrangement of the such as the secretion of inflammatory actin cytoskeletons. MARCKS might, mediators and cytokines t3,'4. therefore, provide a PKC-sensitive reversible crossbridge between the (3) Mitogenesis and transformation. A actin cytoskeleton and the plasma number of neuropeptides that act as growth factors in Swiss 3T3 cells membrane. Such a reversible association of actin is a prerequisite for also induce the phosphorylation of MARCKS18, although it has yet to be directed cellular locomotion during
crosstalk
proven that this event is an intermediate in the mitogenic pathway. A number of reports have suggested that the level of MARCKS is down regulated by transformation 19. A change in cell shape is one of the hallmarks of transformation: cells become much less adherent and round up, there is a marked reduction in the number and thickness of actin stress fibers and the cells become capable of anchorageindependent growth. MARCKS may therefore have a role in maintaining normal cell shape or in delivering anchorage-dependent growth signals, although it is equally possible that its down-regulation during transformation is a consequence of shape change. Profilin Promotion of F-actin stabilization. Profilin is a 12-15 kDa protein that binds monomeric actin and, at high profilin to actin ratios, prevents actin filament formation by monomer sequestration 3,2°,21.
'l'Ca 2+ Cytoskeletal remodeling
Other ADP
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ADP .-"
ATP (~
actin-binding proteins
G-actin Stable actin filaments
Figure 3 Model of how RTKs may regulate the activity of profilin. In unstimulated cells profilin (Prof) binds tightly to phosphatidylinositol 4,5-bisphosphate [Ptdlns(4,5)P2]. This prevents its participation in cytoskeletal remodeling and blocks access of unpflosphorylated phospholipase C5'$ (PLC-71) to Ptdlns(4,5)P 2. Upon addition of EGF, its receptor dimerizes and the activated protein tyrosine kinase (PTK) phosphoryiates itself and tyrosine residues on PLC-~'I. Phosphorylated phospholipase C-~'1(PLC-y-P) can hydrolyse profilin-bound Ptdlns(4,5)P 2 to produce diacylglycerol (DAG), which activates PKC, and inositol trisphosphate (InsP3), which triggers an increase in intracellular Ca2+. The hydrolysis of Ptdlns(4,5)P 2 results in the release of profilin from the membrane allowing it to catalyse adenine nucleotide exchange on G-actin, which promotes stable actin filaments. 441
Biological systems exhibiting transduction crosstalk However, both the cellular levels of profilin, as well as its affinity for actin, appear too low for it to play this role in vivo2°." The role of profilin in vivo is more likely to be related to its capacity to catalyse the exchange of adenine nucleotides on monomeric actin 2°. During actin filament formation, ATP bound to G-actin is hydrolysed to ADP, which remains tightly bound to F-actin22. During filament remodeling, ADP-actin monomers are released. However, while ADP-actin monomers can polymerize, ATP-actin monomers do so more rapidly and form more stable filaments than their dinucleotide-bound counterparts 3,23. Since the exchange of ADP for ATP on G-actin is rate limiting, profilin has the potential to promote actin polymerization and filament stabilization by catalysing actin nucleotide exchange2°. This has been shown in cells that stably overexpress profilin, where actin filament stability, measured by the fluorescence decay of microinjected caged resorufin-actin, is directly proportional to profilin concentration (T. Finkel et al., submitted). However, the observation that microinjected profilin decreases cellular F-actin and prevents the extension of lamellipodia indicates that at high concentrations, profilin can sequester actin monomers in ViVO24. In contrast, the microinjection of highaffinity profilin-actin complexes increase F-actin and enhances membrane ruffling24, consistent with the effect of profilin on actin nucleotide exchange. Profilin binds to polyphosphoinositides.P r o filin binds tightly and specifically to four or five molecules of phosphatidylinositol 4,5-bisphosphate [Ptdlns(4,5)P2] in vitro, and this results in a dissociation of the profilin-actin complex25. Concomitantly, profilin masks PtdIns(4,5)P2 from the hydrolytic action of phospholipase C-7~(PLC-7~)26. Thus, profilin provides a link between a major signal transduction pathway, the phosphatidylinosito] (PtdIns) cycle, and the actin cytoskeleton (Fig. 3). Ultrastructural localization studies demonstrate a significant fraction of profilin in close association with both the internal leaflet of the plasma membrane and external surfaces of intracellular membranes in human leukocytes and platelets 27. This association with the membrane is likely to reflect its direct binding to phosphatidylinositol 4-phosphate [PtdIns4P] and PtdIns(4,5)P2. Stimulation of platelets with thrombin results in rapid PtdIns turnover, the cycling of profilin between the cytosol 442
and the plasma membrane, changes in the levels of the high-affinity profilactin complex and in the polymerization of actin 27. However, it is important to recognize that a number of other actinbinding proteins, including gelsolin, gCap39, severin and protein 4.1 also bind to PtdIns(4,5)P2 (Ref. 28), and any scheme devised to explain the connection of the PtdIns cycle to actin remodeling would also have to account for the actions of these other actinbinding proteins. The receptor tyrosine kinase-profilin connection. As discussed above, PtdIns(4,S)P2 that is bound to profilin is not hydrolysed by PLC-7~26 (Fig. 3). However, when PLC-Tx is phosphorylated by the action of receptor tyrosine kinases (RTKs), such as the receptors for epidermal growth factor (EGF) or platelet derived growth factor (PDGF), it becomes capable of hydrolysing profilinbound Ptdlns(4,5)P2 to generate inosito] trisphosphate [Ins(1,4,5)P3] and diacylglycerol29. This suggests that profilin is now free to interact with actin and stabilize filaments (Fig. 3). It is well known that growth factors such as EGF and PDGF influence the actin cytoskeleton and induce cells to change shape, ruffle their membranes, migrate and divide. However, the mechanism by which RTKs influence the organization of the actin cytoskeleton is unknown. Also obscure is the mechanism by which receptor tyrosine kinases activate inositol 1-phosphate (InslP) turnover, since the phosphorylation of PLC-7~ does not appreciably influence its activity in vitro 2°. Actin-binding proteins such as profilin, which bind to Ptdlns(4,5)P2 and mask it from the action of non-phosphorylated PLC-y1, might be a key intermediate in both these events. It also seems likely that other actin-binding proteins, which bind PtdIns(4,5)P2, such as gelsolin and pCap39, might similarly transduce signals from RTKs to the actin cytoskeleton. An alternative mechanism by which RTKs may influence cytoskeletal events is by direct action of the receptors on actin-binding proteins which bear Src homology 2 (SH2) domains. For example, tensin, an actin-binding protein which is a component of focal contacts, bears an SH2 domain that binds to a number of phosphotyrosine-containing proteins 3°. Tensin may therefore bind directly to autophosphorylated RTKs and mediate their direct association with the actin cytoskeleton. In addition, many RTKs activate phospholipases that generate diacylglycerol and InsP3; these two sec-
TIBS 17 - OCTOBER 1992 ond messengers, in turn, activate PKC and increase intracellular Ca2~. As discussed above, diacylglycerol can nucleate actin filaments 12, Ca 2÷ c a n regulate a large number of actin-binding proteins 1, and PKC can alter actin structure through MARCKS9. The Ras-CAP signal transduction pathway. The Ras family of low molecular weight guanine nucleotide-binding proteins has a role in signal transduction pathways that regulate growth and differentiation31 (also see other reviews in this issue). In the yeast Saccharomyces cerevisiae, Ras activates adenylate cyclase activity in a complex that consists of at least two components: the adenylate cyclase catalytic subunit and an adenylate cyclaseassociated protein (CAP)2°,32. CAP is a bifunctional signal transducer: its aminoterminal domain is necessary and sufficient for cellular responsiveness to activated Ras, and its carboxy-terminal domain has a role in a number of cellular functions including the control of cell morphology. For example, yeast ceils which are null for CAP bud randomly and are defective in actin distribution32o The morphological and nutritional defects associated with the loss of the CAP carboxy-terminal domain are suppressed by overexpression of the profilin gene 32. In addition, cells lacking profilin are abnormally enlarged and round, do not contain actin cables and bud randomly from the cell surface 21, a phenotype similar to that of cells lacking the carboxyterminal domain of CAP32. Mutational analysis and studies using Acanthamoeba profilins indicate that the ability of profilin to suppress the cap- phenotype is dependent on the ability to bind PtdIns(4,5)P2 in conjunction with its actin-binding capacity32. Thus, it appears that CAP and profilin are components of the same signaling pathway, which implies that CAP may be a component of a polyphosphoinositide signal transduction pathway. Alternatively, CAP itself may bind actin, or CAP and profilin may bind to each other. It seems likely that CAP itself is an actinbinding protein, since it is highly homologous to ASP-56, an actin monomer sequestering protein from porcine platelets 33. However, the possibility that CAP may be regulated by phospholipids is of special interest since the activity of Ras has also been shown to be regulated by bioactive phospholipids 34.
Conclusions and perspectives Advances in our understanding of signal transduction and cytoskeletal
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Biological systems exhibiting transduction
structure have allowed a glimpse of the complicated network of events which translates information from the extracellular environment into changes in cellular architecture. Rapid progress is being made in understanding the mechanisms by which individual actin-binding proteins regulat_e actin structure, and are themselves regulated. Much more daunting is the task of understanding how the more than 70 actinbinding proteins interact in vivo. Fortunately, genetic studies of yeast and flies, used in conjunction with the array of techniques already available, are facilitating progress. For example, a recent study demonstrating that the chickadee gene product, required for intercellular cytoplasm transport during Drosophila oogenesis, is homologous to profilin3s, has provided new insight into the regulation and function of actin in vivo.
supported by the NIH, the American Heart Association, and the Cancer Research Institute.
Acknowledgements
I wish to thank P. Goldschmidt for providing me with preprints of unpublished work, and J. Hartwig, P. Janmey, P. Goldschmidt, K. Barker, P. Matsudaira and A. Nairn for discussion and comments on the manuscript. I would also like to thank all the members of my laboratory for helpful discussions and for commenting on the manuscript. Research in the author's laboratory was
References Due to TIBS editorial policy, the number of references cited have been limited. I apologize to those authors whose work has been cited through the reviews of others.
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This article is dedicated to the memory of
~Ef Racker.
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