Cell. Vol. 71, 713-716,
November
27, 1992, Copyright
0
1992 by Cell Press
The MARCKS Brothers: A Family of Protein Kinase C Substrates Alan Aderem
actin-membrane interaction. A detailed understanding of MARCKS structure, of the way MARCKS interacts with calmodulin, actin, and PKC, and of the mechanism by which it cycles between the membrane and cytosol provides a framework for understanding the role of MARCKS in several central biological processes.
Laboratory of Signal Transduction The Rockefeller University New York, New York 10021
The protein kinase C (PKC) family are diacylglycerolactivated, calcium-dependent protein kinases that regulate a large number of cellular responses. An intensive, decade-long research effort has yielded much insight into the structure, catalytic mechanism, and regulation of PKC. More recently, the emphasis has shifted to the identification of physiologically important substrates of PKC, since the characterization of these proteins is essential for an understanding of the mechanism of PKC action. The myristoylated alanine-rich C-kinase substrate, MARCKS, is a widely distributed, specific PKC substrate, whose phosphorylation has been used as a marker of PKC activation in vivo. MARCKS binds both calmodulin and actin (Graff et al., 1989b; Hartwig et al., 1992) and has been implicated in secretion and membrane trafficking, cell motility, the regulation of the cell cycle, and transformation (Wu et al., 1982; Rozengurt et al., 1983; Kligman and Patel, 1986; Blackshear et al., 1986; Wolfman et al., 1987; Aderem et al., 1988; Simek et al., 1989; Thelen et al., 1990). All of these processes necessitate regulated rearrangement of the actin cytoskeleton. Recent data suggest that MARCKS has a role in translating extracellular signals into alterations in actin plasticity and changes in
Mpstoylated Targeting
Membrane Domaln
MacMARCKSIF52
Merbiane DomaIn
Act~n BIndIng? MHZ
KKFSFKKPFKLSGLSFKR
Features
MARCKS is an acidic protein that is unusually rich in alanine, glycine, proline, and glutamic acid. The protein is a rod-shaped molecule containing three distinct domains: an N-terminal myristoylated domain that mediates binding to membranes, a highly conserved MH2 domain of unknown function, and a basic effector domain containing the PKC phosphorylation sites and the calmodulinand actin-binding sites (Figure 1) (Stump0 et al., 1989; Seykora et al., 1991; Harlan et al., 1991; Erusalimsky et al., 1991). The binding of MARCKS to calmodulin and actin is regulated in a complex manner (Graff et al., 1989b; Hartwig et al., 1992). First, MARCKS will bind to calmodulin only in the presence of calcium, and the phosphorylation of MARCKS prevents the binding of calmodulin to it (Graff et al., 1989b). Second, MARCKS binds to the sides of actin filaments and cross-links them, and the cross-linking activity is disrupted by both phosphorylation and calcium-calmodulin (Hartwig et al., 1992). Filament cross-linking requires that MARCKS must either have two actin-binding sites or dimerize. Thus, phosphorylation or calcium-calmodulin either inactivates an actin-binding site or dissociates the dimers.
Structure
of MARCKS
Helical wheel representations of the effector domains show phenylalanine residues that contribute to the amphipathic structure Q, lysine residues that form the calmodulin- and actin-binding sites (+K), and serine residues that are phosphorylated by PKC (‘S).
KRFSFKKSFKLSGFSFKK
MARCKS
Myr,s,ayiated Tarqetlng
Biochemical
Figure 1. The Domain and MacMARCKSIF52
PhOSphO~latlO” Caimod”lln BIndIng ActIn BIndIng MH2
Minireview
-
Cell 714
Figure 2. MARCKS Is Displaced phages When PKC Is Activated
from Punctate
Structures
(A) In quiescent macrophages, MARCKS is located in tures al the substrate-adherent surface of frlopodia. (6) phase photomicrograph. (C)Treatment of macrophages esters results in activatfon of PKC, phosphorylation of its d&placement from the punctate structures. (D) phase photomicrograph.
in Macro-
punctate strucCorrespondrng with phorbol MARCKS, and Correspondfng
The proximity of the phosphorylation sites to the actinand calmodulin-binding sites could explain the reciprocal regulation of phosphorylation and the binding of these two proteins (Figure 1). The effector domain can be modeled as an amphipathic a helix, and the five lysine residues positioned on one side of the helix constitute both the calmodulinand actin-binding sites of MARCKS, hence the competition between these two ligands (Graff et al., 1989b; Hartwig et al., 1992). The three serine residues positioned at the opposite side of the helix are substrates for PKC, and their phosphorylation prevents the binding of calmodulin or actin, presumably because the negative phosphate groups influence the positively charged lysine residues (Figure I). This also explains why calcium-calmodulin prevents the phosphorylation of MARCKS (Graff et al., 1989b). The reciprocal relationship between calmodulin binding and phosphorylation of MARCKS has led to the suggestion that MARCKS functions to sequester calmodulin that could be released upon activation of PKC (Graff et al., 1989b). Localization Targeting of MARCKS to the plasma membrane occurs via its N-terminal, myristoylated membrane-binding domain (Graff et al., 1989a), and this places the substrate in close apposition to PKC (Rosen et al., 1990). PKC-dependent
phosphorylation displaces MARCKS from the membrane, and its subsequent dephosphorylation is accompanied by its reassociation with the membrane (Wang et al., 1989; Thelen et al., 1991). The observation that MARCKS is released from the membrane by the phosphorylation of a site that is remote from the membrane-binding domain (Figure 1) suggests either an allosteric switch mechanism or the existence of additional determinants of membrane association. In macrophages, MARCKS has a punctate distribution, and many of the structures containing MARCKS are found at the substrate-adherent surface of pseudopodia and filopodia (Figure 2; Rosen et al., 1990). Many of these structures also contain vinculin and talin, known components of focal contacts (Rosen et al., 1990). The punctate distribution of vinculin and talin in macrophages contrasts with the plaque-like arrangement (known as focal adhesion plaques), which is typical of adherent fibroblasts (Turner and Burridge, 1991); it has previously been observed in macrophages and at the active edge of fibroblasts during early stages of fibroblast attachment (Marchisio et al., 1987). The colocalization of MARCKS with vinculin and talin at these punctate sites suggests that MARCKS might be located in the initial, more transient adhesion complex that is formed at this site in the locomoting macrophage. lmmunoelectron microscopy demonstrates the presence of MARCKS in clusters at points where actin filaments interact with the cytoplasmic surface of the plasma membrane. Phosphorylation of MARCKS results in its translocation from membrane-bound structures to the cytosol (Figure 2) where it remains closely associated with actin filaments. However, since phosphorylated MARCKS binds to actin but does not cross-link it (Hartwig et al., 1992), these filaments are potentially less rigid. Figure 3 presents a model for MARCKS function that is consistent with current data. It should be noted, however, that much of this model is speculative, since it is mainly derived from in vitro biochemistry. Biological Roles The way in which MARCKS might regulate actin-membrane interactions is suggested by the conditions under which its phosphorylation and synthesis are regulated. A role for MARCKS in motility is suggested by studies using phagocytic cells. In macrophages, MARCKS appears to provide a PKC-sensitive cross-bridge between the actin cytoskeleton and focal contacts at the plasma membrane (Rosen et al., 1990; Figure 2). Such a reversible association of actin is a prerequisite for cellular locomotion. A role for MARCKS in motility is further supported by the observation that, in neutrophils, chemotactic agentscause MARCKS to shuttle between the membrane and the cytosol (Thelen et al., 1991). MARCKS is also highly concentrated in presynaptic junctions and is phosphorylated when synaptosomes are depolarized (Wu et al., 1982; Wang et al., 1989) suggesting a role in secretion or membrane recycling. This is supported by the observation that both tumor necrosis factor and bacterial lipopolysaccharide induce the synthesis of MARCKS and simultaneously prime macrophages and neutrophils for enhanced secretion of inflammatory
Mmireview 715
Figure 3. Model Which MARCKS brane Interaction
lndicatmg a Mechanism by Might Regulate Actin-Mem-
At rest, MARCKS (M) associates with a site on the cytoplasmic face of the membrane, perhaps by bmding to a “receptor” (R). In its nonphosphorylated form, MARCKS cross-links actin into a rigid meshwork at the membrane (mem actin, cross-hatched). An agonist receptor activates PKC through a cascade involving ) , d\ G proteins (G) and phospholipase C (PLC). f ) PKC phosphorylates MARCKS, which is released from the membrane. Phosphorylated cytosolic MARCKS remains associated with Phosphatase; actin filaments but cannot cross-link actin. The i\ actin linked to phosphorylated MARCKS may +--be spatially separated from the membrane and -PI \ j._ more plastic (cyt actm, hatched). When LI P d’ MARCKS is dephosphorylated, it returns to the / I,MI w membrane and again cross-links actin. \/I An increase in intracellular calcium proG2 motes the binding of calmodulin (Cal) to MARCKS, thereby inhibiting its actin cross-linking activity. This results in a less rigid actin structure, still linked through MARCKS to the membrane (mem actln, hatched). A decrease In intracellular calcium shifts the equilibrium to the resting state, in which MARCKS cross-links actin at the membrane (mem actin, cross-hatched). Since calcium levels are known to oscillate following cellular stimulation, MARCKS would mediate cycles of calcium-dependent actin cross-lmkmg activity at the membrane. Upon phosphorylation, MARCKS is unable to bind calmodulm and is released from the membrane, resulting in local destabilization of the actin skeleton (cyi actin, hatched).
mediators and cytokines (Aderem et al., 1988; Thelen et al., 1990). Several neuropeptides that act as growth factors in Swiss 3T3 cells induce the phosphorylation of MARCKS (Rozengurt et al., 1983), although it has not yet been proven that this event is an intermediate in the mitogenic pathway. Recently, it has been shown that a variety of mitogenic agents decrease MARCKS expression, leading to the suggestion that MARCKS down-regulation might be required for reentry of cells into the cell cycle (Brooks et al., 1992). MARCKS expression is also down-regulated during cellular transformation (Wolfman et al., 1987). Transformation is accompanied by a marked change in cell shape, and it is tempting to speculate that MARCKS has a role in maintaining normal cell shape or in delivering anchorage-dependent growth signals. However, it is equally possible that the down-regulation of MARCKS during transformation is a consequence of shape change.
MacMA RCKSlF52 MacMARCKS/F52, a second member of the MARCKS family of PKC substrates, is highly enriched in macrophages following stimulation with lipopolysaccharide (Umekage and Kato, 1991; Blackshear et al., 1992; Li and Aderem, 1992). It has an amino acid composition and domain structure similar to MARCKS (Figure l), and phosphorylation also regulates its capacity to bind calmodulin (Blackshear et al., 1992; Li and Aderem, 1992). The N-terminal myristoylation domain of MacMARCKS/F52 is subtly different than that of MARCKS, and preliminary data suggest that MacMARCKSIF52 is targeted to a different subcellular location than MARCKS. This is reminiscent of the Ras family of small GTP-binding proteins, in which a similar effector domain is targeted to distinct membrane loci by small differences in the C-terminus. MacMARCKSIF52
has
an almost
identical
effector
do-
main to MARCKS, except that the second phosphorylatable serine of MARCKS is replaced by proline in MacMARCKSIF52 (Figure 1). It is unlikely that this proline residue disrupts the proposed a helix, because MacMARCKSIF52 binds calmodulin and is phosphorylated by PKC (Blackshear et al., 1992; Li and Aderem, 1992). Considering the similarity between the effector domains of MARCKS and MacMARCKSIF52, it will be interesting to determine whether MacMARCKS/F52 also binds actin. The myristoylation domains of both MacMARCKSIF52 and MARCKS are separated from their common effector domain by a highly conserved MH2 domain (Figure 1). Interestingly, the mannose-6-phosphate/insulin-like growth factor II receptor contains a region apparently involved in internalization that is homologous to the MH2 domain (Li and Aderem, 1992; Blackshear et al., 1992). The MH2 domain may also interact with actin or contain a dimerization motif, since the actin cross-linking activity of MARCKS implies that MARCKS either contains a second actinbinding site or dimerizes (Hartwig et al., 1992). Since an intron is located within the MH2 domain, it has also been suggested that the RNA encoding this domain may have a role in directing RNA splicing (Stump0 et al., 1989; Harlan et al., 1991).
Other MA RCKS-Related
Proteins
MARCKS and MacMARCKSIF52 bear strong functional similarity to a neurospecific PKC substrate known as GAP43 or neuromodulin (reviewed in Gordon-Weeks, 1989). GAP-43, like MARCKS and MacMARCKS/F52, is an acidic rod-shaped protein with a predominance of alanine, glytine, proline, and glutamic acid. GAP-43 also contains a basic phosphorylation domain that includes its calmodulin-binding site; phosphorylation also prevents calmodulin binding. However, in contrast to MARCKS and MacMARCKSIF52, GAP-43 associates with calmodulin in the
Cell 716
absence of calcium. In addition, GAP-43 is palmitoylated rather than myristoylated at its N-terminus. Both MARCKS and GAP-43 appear to have a role in regulating plasticity of the cytoskeleton: GAP-43 associates with the actin cytoskeleton in neuronal growth cones (Gordon-Weeks, 1989). Adducin, a PKC substrate that promotes the association of actin with spectrin in a calmodulin-regulated manner, has both structural and functional similarities to MARCKS and MacMAFiCKUF52 (Joshi et al., 1991). Adducin is composed of highly homologous a and 8 subunits; both subunits contain identical stretches of 22 amino acids in their C-termini with sequence similarity to the effector domains of MARCKS and MacMARCKSIF52 (Joshi et al., 1991). The C-termini of the a and 5 subunits of adducin also bear the PKC phosphorylation sites and the domain that binds calcium-calmodulin (Joshi et al., 1991). It is therefore possible that this motif in the effector domain of MARCKS serves a common function in regulating calmodulin binding and perhaps actin binding in a diverse array of cytoskeletal proteins. Conclusions MARCKS and MacMARCKS/F52 are members of a family of myristoylated PKC substrates that bind calmodulin in a calcium-dependent and phosphorylation-regulated manner. The two proteins are differentially expressed, suggesting that they have different functions. MARCKS is widely distributed, while MacMARCKSF52 is restricted in expression and is highly enriched in macrophages. The MARCKS proteins have three domains: a membranebinding domain, an MH2 domain, and an effector domain that binds calmodulin and actin. Two other calmodulinbinding PKC substrates that also have a role in regulating the actin cytoskeleton, GAP-43 and adducin, have similarities to the MARCKS family. The data suggest the existence of an extended family of PKC substrates that are targeted to different subcellular locations and that function to integrate PKC and calcium-calmodulin-dependent signals in the control of the actin cytoskeleton References Aderem, A. A., Albert, K. A., Keum, P., and Cohn, Z. A. (1988). Nature
M. M., Wang, J. K. T., Greengard, 332, 362-364.
Blackshear, Biol. Chem.
B. P., and Witters,
P. J., Wen, L., Glynn, 267, 1459-1469.
L. A. (1986).
Blackshear, P. J., Verghese, G. M., Johnson, J. D., Haupt, Stumpo, D. J. (1992). J. Biol. Chem. 267, 13540-13546. Brooks, S. F., Herget, T., Broad, Chem. 267, 14212-14218.
S., and Rozengurt,
Erusalimsky, J. D., Brooks, S. F., Herget, T., Morris, E. (1991). J. Biol. Chem. 266, 7073-7080. Gordon-Weeks,
P. R. (1989).
Graft, J. M., Gordon, 503-506.
Trends
Neuroscr.
J. I,, and Blackshear,
J.
D. M., and
E. (1992). J. Biol. C., and Rozengurt,
72, 363-365.
P. J. (1989a).
Science
Graff, J. M., Young, T. N., Johnson, J. D., and Blackshear, (1989b). J. Biol. Chem. 264, 21818-21823.
246, P. J.
Harlan, D. M., Graff, J. M., Stumpo, D. J., Eddy, R. L., Jr., Shows, T. B., Boyle, J. M., and Blackshear, P. J. (1991). J. Biol. Chem. 266, 14399-14405. Hartwig, Aderem,
J. H., Thelen, M., Rosen, A., Janmey, A. (1992). Nature 356, 618-622.
P. A., Nairn. A. C., and
Joshi, R., Gilligan, D. M., Otto, E., McLaughlin, (1991). J. Cell Biol. 775, 665-675. Klrgman.
D., and Patel, J. (1986).
LI, J., and Aderem, Marchisio, G. (1987). Rosen, (1990).
A. (1992).
T, and Bennett,
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47, 298-303.
Cell 70, 791-801.
P. C., Cirillo, D.,Teti, A., Zambonin-Zallone, Exp. Cell Res. 769, 202-214.
A., andTarone,
A , Keenan, K. F., Thelen, M., Nairn, A. C J. Exp. Med. 772, 1211-1215.
Rozengurt, Natl. Acad.
V.
and Aderem,
E.. Rodriguez-Pena, M., and Smith, Sci. USA 80, 7244-7248.
Seykora, J. T., Ravetch, J.V., andAderem,A. Sci. USA 88, 2505-2509.
K. A. (1983).
A A. Proc.
(1991). Proc. Natl. Acad.
Srmek, S. L., Kligman, D., Patel, J., and Colburn, Natl. Acad. Sci. USA 86, 7410-7414.
N. H. (1989)
Proc.
Stumpo, D. J., Graff, J. M.. Albert, K. A., Greengard, P., and Blackshear, P. J. (1989). Proc. Natl. Acad. Sci. USA 86, 4012-4016. Thelen, M., Rosen, A., Nairn, A. C., and Aderem, Acad. Sci. USA 87, 5603-5607. Thelen. M., Rosen, 357, 320-322. Turner, 853. Umekage,
A., Narrn,
C. E., and Burridge, T , and Kato,
A. (1990). Proc.
A. C., and Aderem, K. (1991).
K. (1991).
Curr.
FEBS
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Lett. 286, 147-151.
Wang, J. K. T., Walaas, S. I., Sihra, T. S., Aderem, A., and Greengard, P. (1989). Proc. Natl. Acad. Sci. USA 86, 2253-2256. Wolfman, A., Wingrove, T. G., Blackshear, (1987). J. Biol. Chem. 262, 16546-16552.
P. J., and Macara,
Wu, W. S., Walaas, S. I., Nairn, A. C., and Greengard, Natl. Acad. Sci. USA 79, 5249-5253.
P. (1982).
I. G. Proc.
November
27, 1992, Copyright
0
1992 by Cell Press
The MARCKS Brothers: A Family of Protein Kinase C Substrates Alan Aderem
actin-membrane interaction. A detailed understanding of MARCKS structure, of the way MARCKS interacts with calmodulin, actin, and PKC, and of the mechanism by which it cycles between the membrane and cytosol provides a framework for understanding the role of MARCKS in several central biological processes.
Laboratory of Signal Transduction The Rockefeller University New York, New York 10021
The protein kinase C (PKC) family are diacylglycerolactivated, calcium-dependent protein kinases that regulate a large number of cellular responses. An intensive, decade-long research effort has yielded much insight into the structure, catalytic mechanism, and regulation of PKC. More recently, the emphasis has shifted to the identification of physiologically important substrates of PKC, since the characterization of these proteins is essential for an understanding of the mechanism of PKC action. The myristoylated alanine-rich C-kinase substrate, MARCKS, is a widely distributed, specific PKC substrate, whose phosphorylation has been used as a marker of PKC activation in vivo. MARCKS binds both calmodulin and actin (Graff et al., 1989b; Hartwig et al., 1992) and has been implicated in secretion and membrane trafficking, cell motility, the regulation of the cell cycle, and transformation (Wu et al., 1982; Rozengurt et al., 1983; Kligman and Patel, 1986; Blackshear et al., 1986; Wolfman et al., 1987; Aderem et al., 1988; Simek et al., 1989; Thelen et al., 1990). All of these processes necessitate regulated rearrangement of the actin cytoskeleton. Recent data suggest that MARCKS has a role in translating extracellular signals into alterations in actin plasticity and changes in
Mpstoylated Targeting
Membrane Domaln
MacMARCKSIF52
Merbiane DomaIn
Act~n BIndIng? MHZ
KKFSFKKPFKLSGLSFKR
Features
MARCKS is an acidic protein that is unusually rich in alanine, glycine, proline, and glutamic acid. The protein is a rod-shaped molecule containing three distinct domains: an N-terminal myristoylated domain that mediates binding to membranes, a highly conserved MH2 domain of unknown function, and a basic effector domain containing the PKC phosphorylation sites and the calmodulinand actin-binding sites (Figure 1) (Stump0 et al., 1989; Seykora et al., 1991; Harlan et al., 1991; Erusalimsky et al., 1991). The binding of MARCKS to calmodulin and actin is regulated in a complex manner (Graff et al., 1989b; Hartwig et al., 1992). First, MARCKS will bind to calmodulin only in the presence of calcium, and the phosphorylation of MARCKS prevents the binding of calmodulin to it (Graff et al., 1989b). Second, MARCKS binds to the sides of actin filaments and cross-links them, and the cross-linking activity is disrupted by both phosphorylation and calcium-calmodulin (Hartwig et al., 1992). Filament cross-linking requires that MARCKS must either have two actin-binding sites or dimerize. Thus, phosphorylation or calcium-calmodulin either inactivates an actin-binding site or dissociates the dimers.
Structure
of MARCKS
Helical wheel representations of the effector domains show phenylalanine residues that contribute to the amphipathic structure Q, lysine residues that form the calmodulin- and actin-binding sites (+K), and serine residues that are phosphorylated by PKC (‘S).
KRFSFKKSFKLSGFSFKK
MARCKS
Myr,s,ayiated Tarqetlng
Biochemical
Figure 1. The Domain and MacMARCKSIF52
PhOSphO~latlO” Caimod”lln BIndIng ActIn BIndIng MH2
Minireview
-
Cell 714
Figure 2. MARCKS Is Displaced phages When PKC Is Activated
from Punctate
Structures
(A) In quiescent macrophages, MARCKS is located in tures al the substrate-adherent surface of frlopodia. (6) phase photomicrograph. (C)Treatment of macrophages esters results in activatfon of PKC, phosphorylation of its d&placement from the punctate structures. (D) phase photomicrograph.
in Macro-
punctate strucCorrespondrng with phorbol MARCKS, and Correspondfng
The proximity of the phosphorylation sites to the actinand calmodulin-binding sites could explain the reciprocal regulation of phosphorylation and the binding of these two proteins (Figure 1). The effector domain can be modeled as an amphipathic a helix, and the five lysine residues positioned on one side of the helix constitute both the calmodulinand actin-binding sites of MARCKS, hence the competition between these two ligands (Graff et al., 1989b; Hartwig et al., 1992). The three serine residues positioned at the opposite side of the helix are substrates for PKC, and their phosphorylation prevents the binding of calmodulin or actin, presumably because the negative phosphate groups influence the positively charged lysine residues (Figure I). This also explains why calcium-calmodulin prevents the phosphorylation of MARCKS (Graff et al., 1989b). The reciprocal relationship between calmodulin binding and phosphorylation of MARCKS has led to the suggestion that MARCKS functions to sequester calmodulin that could be released upon activation of PKC (Graff et al., 1989b). Localization Targeting of MARCKS to the plasma membrane occurs via its N-terminal, myristoylated membrane-binding domain (Graff et al., 1989a), and this places the substrate in close apposition to PKC (Rosen et al., 1990). PKC-dependent
phosphorylation displaces MARCKS from the membrane, and its subsequent dephosphorylation is accompanied by its reassociation with the membrane (Wang et al., 1989; Thelen et al., 1991). The observation that MARCKS is released from the membrane by the phosphorylation of a site that is remote from the membrane-binding domain (Figure 1) suggests either an allosteric switch mechanism or the existence of additional determinants of membrane association. In macrophages, MARCKS has a punctate distribution, and many of the structures containing MARCKS are found at the substrate-adherent surface of pseudopodia and filopodia (Figure 2; Rosen et al., 1990). Many of these structures also contain vinculin and talin, known components of focal contacts (Rosen et al., 1990). The punctate distribution of vinculin and talin in macrophages contrasts with the plaque-like arrangement (known as focal adhesion plaques), which is typical of adherent fibroblasts (Turner and Burridge, 1991); it has previously been observed in macrophages and at the active edge of fibroblasts during early stages of fibroblast attachment (Marchisio et al., 1987). The colocalization of MARCKS with vinculin and talin at these punctate sites suggests that MARCKS might be located in the initial, more transient adhesion complex that is formed at this site in the locomoting macrophage. lmmunoelectron microscopy demonstrates the presence of MARCKS in clusters at points where actin filaments interact with the cytoplasmic surface of the plasma membrane. Phosphorylation of MARCKS results in its translocation from membrane-bound structures to the cytosol (Figure 2) where it remains closely associated with actin filaments. However, since phosphorylated MARCKS binds to actin but does not cross-link it (Hartwig et al., 1992), these filaments are potentially less rigid. Figure 3 presents a model for MARCKS function that is consistent with current data. It should be noted, however, that much of this model is speculative, since it is mainly derived from in vitro biochemistry. Biological Roles The way in which MARCKS might regulate actin-membrane interactions is suggested by the conditions under which its phosphorylation and synthesis are regulated. A role for MARCKS in motility is suggested by studies using phagocytic cells. In macrophages, MARCKS appears to provide a PKC-sensitive cross-bridge between the actin cytoskeleton and focal contacts at the plasma membrane (Rosen et al., 1990; Figure 2). Such a reversible association of actin is a prerequisite for cellular locomotion. A role for MARCKS in motility is further supported by the observation that, in neutrophils, chemotactic agentscause MARCKS to shuttle between the membrane and the cytosol (Thelen et al., 1991). MARCKS is also highly concentrated in presynaptic junctions and is phosphorylated when synaptosomes are depolarized (Wu et al., 1982; Wang et al., 1989) suggesting a role in secretion or membrane recycling. This is supported by the observation that both tumor necrosis factor and bacterial lipopolysaccharide induce the synthesis of MARCKS and simultaneously prime macrophages and neutrophils for enhanced secretion of inflammatory
Mmireview 715
Figure 3. Model Which MARCKS brane Interaction
lndicatmg a Mechanism by Might Regulate Actin-Mem-
At rest, MARCKS (M) associates with a site on the cytoplasmic face of the membrane, perhaps by bmding to a “receptor” (R). In its nonphosphorylated form, MARCKS cross-links actin into a rigid meshwork at the membrane (mem actin, cross-hatched). An agonist receptor activates PKC through a cascade involving ) , d\ G proteins (G) and phospholipase C (PLC). f ) PKC phosphorylates MARCKS, which is released from the membrane. Phosphorylated cytosolic MARCKS remains associated with Phosphatase; actin filaments but cannot cross-link actin. The i\ actin linked to phosphorylated MARCKS may +--be spatially separated from the membrane and -PI \ j._ more plastic (cyt actm, hatched). When LI P d’ MARCKS is dephosphorylated, it returns to the / I,MI w membrane and again cross-links actin. \/I An increase in intracellular calcium proG2 motes the binding of calmodulin (Cal) to MARCKS, thereby inhibiting its actin cross-linking activity. This results in a less rigid actin structure, still linked through MARCKS to the membrane (mem actln, hatched). A decrease In intracellular calcium shifts the equilibrium to the resting state, in which MARCKS cross-links actin at the membrane (mem actin, cross-hatched). Since calcium levels are known to oscillate following cellular stimulation, MARCKS would mediate cycles of calcium-dependent actin cross-lmkmg activity at the membrane. Upon phosphorylation, MARCKS is unable to bind calmodulm and is released from the membrane, resulting in local destabilization of the actin skeleton (cyi actin, hatched).
mediators and cytokines (Aderem et al., 1988; Thelen et al., 1990). Several neuropeptides that act as growth factors in Swiss 3T3 cells induce the phosphorylation of MARCKS (Rozengurt et al., 1983), although it has not yet been proven that this event is an intermediate in the mitogenic pathway. Recently, it has been shown that a variety of mitogenic agents decrease MARCKS expression, leading to the suggestion that MARCKS down-regulation might be required for reentry of cells into the cell cycle (Brooks et al., 1992). MARCKS expression is also down-regulated during cellular transformation (Wolfman et al., 1987). Transformation is accompanied by a marked change in cell shape, and it is tempting to speculate that MARCKS has a role in maintaining normal cell shape or in delivering anchorage-dependent growth signals. However, it is equally possible that the down-regulation of MARCKS during transformation is a consequence of shape change.
MacMA RCKSlF52 MacMARCKS/F52, a second member of the MARCKS family of PKC substrates, is highly enriched in macrophages following stimulation with lipopolysaccharide (Umekage and Kato, 1991; Blackshear et al., 1992; Li and Aderem, 1992). It has an amino acid composition and domain structure similar to MARCKS (Figure l), and phosphorylation also regulates its capacity to bind calmodulin (Blackshear et al., 1992; Li and Aderem, 1992). The N-terminal myristoylation domain of MacMARCKS/F52 is subtly different than that of MARCKS, and preliminary data suggest that MacMARCKSIF52 is targeted to a different subcellular location than MARCKS. This is reminiscent of the Ras family of small GTP-binding proteins, in which a similar effector domain is targeted to distinct membrane loci by small differences in the C-terminus. MacMARCKSIF52
has
an almost
identical
effector
do-
main to MARCKS, except that the second phosphorylatable serine of MARCKS is replaced by proline in MacMARCKSIF52 (Figure 1). It is unlikely that this proline residue disrupts the proposed a helix, because MacMARCKSIF52 binds calmodulin and is phosphorylated by PKC (Blackshear et al., 1992; Li and Aderem, 1992). Considering the similarity between the effector domains of MARCKS and MacMARCKSIF52, it will be interesting to determine whether MacMARCKS/F52 also binds actin. The myristoylation domains of both MacMARCKSIF52 and MARCKS are separated from their common effector domain by a highly conserved MH2 domain (Figure 1). Interestingly, the mannose-6-phosphate/insulin-like growth factor II receptor contains a region apparently involved in internalization that is homologous to the MH2 domain (Li and Aderem, 1992; Blackshear et al., 1992). The MH2 domain may also interact with actin or contain a dimerization motif, since the actin cross-linking activity of MARCKS implies that MARCKS either contains a second actinbinding site or dimerizes (Hartwig et al., 1992). Since an intron is located within the MH2 domain, it has also been suggested that the RNA encoding this domain may have a role in directing RNA splicing (Stump0 et al., 1989; Harlan et al., 1991).
Other MA RCKS-Related
Proteins
MARCKS and MacMARCKSIF52 bear strong functional similarity to a neurospecific PKC substrate known as GAP43 or neuromodulin (reviewed in Gordon-Weeks, 1989). GAP-43, like MARCKS and MacMARCKS/F52, is an acidic rod-shaped protein with a predominance of alanine, glytine, proline, and glutamic acid. GAP-43 also contains a basic phosphorylation domain that includes its calmodulin-binding site; phosphorylation also prevents calmodulin binding. However, in contrast to MARCKS and MacMARCKSIF52, GAP-43 associates with calmodulin in the
Cell 716
absence of calcium. In addition, GAP-43 is palmitoylated rather than myristoylated at its N-terminus. Both MARCKS and GAP-43 appear to have a role in regulating plasticity of the cytoskeleton: GAP-43 associates with the actin cytoskeleton in neuronal growth cones (Gordon-Weeks, 1989). Adducin, a PKC substrate that promotes the association of actin with spectrin in a calmodulin-regulated manner, has both structural and functional similarities to MARCKS and MacMAFiCKUF52 (Joshi et al., 1991). Adducin is composed of highly homologous a and 8 subunits; both subunits contain identical stretches of 22 amino acids in their C-termini with sequence similarity to the effector domains of MARCKS and MacMARCKSIF52 (Joshi et al., 1991). The C-termini of the a and 5 subunits of adducin also bear the PKC phosphorylation sites and the domain that binds calcium-calmodulin (Joshi et al., 1991). It is therefore possible that this motif in the effector domain of MARCKS serves a common function in regulating calmodulin binding and perhaps actin binding in a diverse array of cytoskeletal proteins. Conclusions MARCKS and MacMARCKS/F52 are members of a family of myristoylated PKC substrates that bind calmodulin in a calcium-dependent and phosphorylation-regulated manner. The two proteins are differentially expressed, suggesting that they have different functions. MARCKS is widely distributed, while MacMARCKSF52 is restricted in expression and is highly enriched in macrophages. The MARCKS proteins have three domains: a membranebinding domain, an MH2 domain, and an effector domain that binds calmodulin and actin. Two other calmodulinbinding PKC substrates that also have a role in regulating the actin cytoskeleton, GAP-43 and adducin, have similarities to the MARCKS family. The data suggest the existence of an extended family of PKC substrates that are targeted to different subcellular locations and that function to integrate PKC and calcium-calmodulin-dependent signals in the control of the actin cytoskeleton References Aderem, A. A., Albert, K. A., Keum, P., and Cohn, Z. A. (1988). Nature
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