- Email: [email protected]
Protein Kinase C Regulation: C1 Meets C-tail
Structure
Previews Garneau, J.E., Dupuis, M.E., Villion, M., Romero, D.A., Barrangou, R., Boyaval, P., Fremaux, C., Horvath, P., Magadan, A.H., and Moineau, S. (2010). Nature 468, 67–71. Haft, D., Selengut, J., Mongodin, E., and Nelson, K. (2005). PLoS Comput. Biol. 1, e60. Hale, C.R., Zhao, P., Olson, S., Duff, M.O., Graveley, B.R., Wells, L., Terns, R.M., and Terns, M.P. (2009). Cell 139, 945–956.
Haurwitz, R.E., Jinek, M., Wiedenheft, B., Zhou, K., and Doudna, J.A. (2010). Science 329, 1355–1358.
Marraffini, L.A., and Sontheimer, E.J. (2008). Science 322, 1843–1845.
Horvath, P., and Barrangou, R. (2010). Science 327, 167–170.
Mojica, F.J., Diez-Villasenor, C., Soria, E., and Juez, G. (2000). Mol. Microbiol. 36, 244–246.
Kunin, V., Sorek, R., and Hugenholtz, P. (2007). Genome Biol. 8, R61. Makarova, K.S., Grishin, N.V., Shabalina, S.A., Wolf, Y.I., and Koonin, E.V. (2006). Biol. Direct 1, 7.
Sorek, R., Kunin, V., and Hugenholtz, P. (2008). Nat. Rev. Microbiol. 6, 181–186. Wang, R., Preamplume, G., Terns, M.P., Terns, R.M., and Li, H. (2011). Structure 19, this issue, 257–264.
Protein Kinase C Regulation: C1 Meets C-tail Marcelo G. Kazanietz1,* and Mark A. Lemmon2,* 1Department
of Pharmacology of Biochemistry and Biophysics University of Pennsylvania School of Medicine, Philadelphia, PA, USA *Correspondence: [email protected] (M.G.K.), [email protected] (M.A.L.) DOI 10.1016/j.str.2011.01.004 2Department
In a recent issue of Cell, Leonard et al. (2011) describe the structure of PKCbII, an AGC kinase, revealing an unanticipated intramolecular autoinhibitory interaction between its C-terminal tail and the diacylglycerol and phorbol ester binding site of its C1b domain.
The three-dimensional structure of an intact protein kinase C (PKC), an archetypal lipid membrane effector, has long been anticipated. Following determination of the kinase domain structures for several AGC family Ser-Thr kinases (named after prominent members of the families PKA, PKG, and PKC), key questions have focused on how the kinase domain is regulated by other domains in these tightly controlled modular proteins. In the January 7 issue of Cell, Leonard et al. (2011) describe an important step toward this goal, with a 4 A˚ resolution structure of PKCbII that contains almost 80% of the intact protein–lacking only one of the regulatory domains. The new structure, supported by biochemical and small-angle X-ray scattering studies, adds an important new twist to our understanding of the multistep regulation of PKC. PKC isozymes are widely implicated in human physiology and disease, and their involvement in cancer progression is well established. PKCs are described as classical (cPKCa, bI, bII, and g), novel (nPKCd, 3, h, and q), or atypical (aPKCz and l/i) based on their biochemical requirements
for activation. Only cPKCs respond to calcium, whereas both cPKCs and nPKCs are sensitive to phorbol esters and the lipid second messenger diacylglycerol (DAG). Activation of PKCs is well known to proceed through multiple steps, the first of which involves transphosphorylation (required for maturation to an activatable state) and autophosphorylation. Even once ‘‘primed’’ by phosphorylation, PKC isoforms remain ‘‘autoinhibited’’ by intramolecular interactions between the N-terminal pseudosubstrate region and the active site of the C-terminal kinase domain (Figure 1A). Relieving this autoinhibition also involves multiple steps. In cPKCs, the C2 domain first binds Ca2+ and phospholipids, targeting the enzyme to the membrane. Once membrane-associated, binding of the C1 domains to DAG is thought to reverse autoinhibition by the pseudosubstrate region, leading to full activation of the enzyme (Newton, 2009). Now, the work from Hurley’s group yields two major new findings. First, the structure reveals an additional step in the allosteric activation of PKCbII. The kinase domain appears to be maintained in an inactive state by unexpected intra-
144 Structure 19, February 9, 2011 ª2011 Elsevier Ltd All rights reserved
molecular autoinhibitory interactions between the regulatory C1b domain and an ‘‘NFD motif’’ in the C-terminal tail. Second, the work argues that the two C1 domains in PKCbII (C1a and C1b) play substantially different roles, as predicted from several biochemical and pharmacological studies (Colon-Gonzalez and Kazanietz, 2006) (see below). As with all AGC kinases, the C-terminal tail of PKCbII (residues 621-673) is an important cis-acting regulatory module, wrapping around the N-lobe of the kinase domain (Figure 1B) to stabilize its active conformation (Pearce et al., 2010). The hydrophobic motif in the C-tail (residues 656-661) helps position the key aC helix in the active structure, and the flexible ‘‘turn motif’’ (residues 624-650) contributes to the ATP binding site (Grodsky et al., 2006). In PKCbII, phosphorylation of these regulatory elements promotes their ability to stabilize the active kinase. Just as these C-tail interactions are crucial for stabilizing the active kinase, they also provide vulnerability that seems to be exploited in PKCbII regulation. The structure reported by Leonard et al. (2011) reveals an intriguing intramolecular
Structure
Previews A
C-tail
B
C1b N-lobe ATP
NFD helix
F629 C-lobe
C-tail
C-tail
C C1b
N ATP
N
F629
F629
-P
C
active
NFD helix
ATP
-P
C
autoinhibited
Figure 1. PKCbII Autoinhibition (A) Domain composition of PKCbII. PS designates the pseudosubstrate region. The C-terminal tail is colored red. (B) Structure of kinase domain and C1b domain regions of PKCbII (from PDB ID 3PFQ). Kinase domain is gray, C-tail is red, and C1b domain is cyan. F629 in the NFD motif is contained within a helix that limits its access to the ATP binding site. The NFD helix and adjacent sequences project into the C1b DAG binding site. (C) Cartoon representation of PKCbII autoinhibition. Without C1b domain interaction, the NFD helix is ‘‘unwound,’’ and F629 can contribute to the ATP binding site. The intramolecular interaction with the C1b domain stabilizes helix formation in the NFD motif, rotating F629 out of the ATP binding site, leading to inhibition of the kinase.
interaction in which the C1b domain of PKCbII docks onto a binding site comprising residues 619-633 from the regulatory C-tail plus conserved elements in the kinase domain N-lobe. Moreover, it is the DAG binding site of the C1b domain that mediates this intramolecular contact. Although intramolecular interactions between the C1 domains and the rest of PKCbII might have been expected, their
consequence for the conformation of the kinase domain was not. The region of the C-tail that interacts with the C1b domain is one that alternatively plays an important role in stabilizing the active site of the kinase domain, and has been termed the ‘‘active-site tether’’ (Kannan et al., 2007). In protein kinase A (PKA), the side chain of a conserved phenylalanine (F327) in this region projects into
the ATP-binding pocket and contacts the bound nucleotide. In the PKCbII structure, the corresponding phenylalanine (F629) instead contacts the C1b domain and is prevented from participating in ATP binding (Figure 1B). This key phenylalanine lies in what Hurley and coworkers call the ‘‘NFD motif.’’ The NFD motif is in a region previously noted to be flexible in PKCbII, and forms an a helix both in the new PKCbII structure and in a structure of the isolated inhibitor-bound PKCbII kinase domain (Grodsky et al., 2006). By contrast, this helix is not seen in an ATPbound active PKC kinase domain structure (Takimura et al., 2010), where the NFD motif phenylalanine is freed to project into the ATP-binding site (Figure 1C). These structural details suggest an attractive model. In the absence of DAG, PKCbII is subject to at least two modes of autoinhibition. One involves the pseudosubstrate region. The second arises because the DAG-binding site of the C1b domain associates with the NFD motif in the regulatory C-tail, and ‘‘clamps’’ it in a position that is suboptimal for kinase activity (Figure 1C). In doing so, an a helix is formed in the NFD region, which appears to be characteristic of the inactive state. Only when the C1b domain is engaged by DAG (or phorbol esters) is this clamp released (and the NFD helix unfolded), allowing the kinase to adopt its fully active configuration. Other autoinhibitory sites may also need to be released, such as those involving isozyme-specific motifs required for intracellular compartmentalization (Stebbins and Mochly-Rosen, 2001).
The C1 Domain: More than a Lipid-Binding Motif DAG or phorbol ester binding to C1 domains confers a high-affinity interaction between PKC and the membrane. Tandem C1 domains are found in cPKCs, nPKCs, and other kinases (protein kinase D isozymes and DAG kinases). Other signaling proteins have only one C1 domain, including MRCK (myotonic dystrophy kinase-related Cdc42 binding kinase), Munc-13 scaffolding proteins, and small G protein regulators such as RasGRPs (guanine nucleotide exchangers) or the a- and b-chimaerins (Rac GTPase activating proteins, or RacGAPs).
Structure 19, February 9, 2011 ª2011 Elsevier Ltd All rights reserved 145
Structure
Previews As mentioned above, a major suggestion of the Hurley paper is that the C1a and C1b domains in PKCbII have distinct roles. Indeed, the functional nonequivalence of C1a and C1b became evident in early analyses of membrane translocation of C1 domain mutants and later from biochemical studies (Colon-Gonzalez and Kazanietz, 2006). Despite significant sequence homology, individual C1 domains also differ in their ability to bind ligands and penetrate membranes (Colon-Gonzalez and Kazanietz, 2006). According to the model postulated by Hurley and coworkers, the C1a domain of PKCbII is exposed and is the first to bind DAG in the membrane after initial priming of PKC by calcium. This step may provide the energy for disrupting autoinhibitory interactions between the N-terminal pseudosubstrate region and the kinase domain. Full kinase activation then occurs only after the C1b clamp is released from the NFD motif, coincident with its association with DAG. Despite the obvious differences between PKCs and proteins that contain a single C1 domain, there are similarities between the mechanisms of regulation of PKCbII and b2-chimaerin, for example. In both cases, the ‘‘closed’’ conformation is stabilized by C1 domain association with other domains in the same protein, with which DAG/phorbol ester and phospholipid binding must compete. Like the C1b domain in the Hurley PKCbII structure, the C1 domain in b2-chimaerin is buried, contacting the catalytic (Rac-GAP) domain and other regions. Disrupting the intramolecular C1 domain
interactions by mutagenesis renders b2-chimaerin more sensitive to phorbol 12-myristate 13-acetate (PMA)-induced translocation to membranes and confers elevated Rac-GAP activity (Canagarajah et al., 2004). Leonard et al. (2011) see a similar effect when they disrupt the crystallographically observed clamp in PKCbII. Interestingly, the chimaerins require a higher concentration of PMA for translocation than PKCs do, possibly reflecting a more extensive C1 domain burial. However, in response to physiological stimuli such as EGF, which generates DAG through activation of phospholipase C, full translocation of chimaerins is observed despite the lower affinity of C1 domains for DAG than for PMA (Colon-Gonzalez et al., 2008). DAG binding to the C1 domain is required, but not sufficient, for chimaerin translocation, suggesting that other factors contribute to disruption of the intramolecular autoinhibitory interactions. One possibility is that association of other proteins with the chimaerin N-terminal region weakens intramolecular C1 domain interactions. Interaction of PKCs with other proteins appears crucial for dictating the distinctive intracellular localization of each isoform, and thus their differential access to substrates. The C1 domains may play a role in this. Indeed, both C1a and C1b domains have been shown to engage in protein-protein interactions, one key example being the association of p23/Tmp21 with the C1b domain of nPKCs, anchoring them in the perinuclear region (Colon-Gonzalez and Kazanietz, 2006).
146 Structure 19, February 9, 2011 ª2011 Elsevier Ltd All rights reserved
It remains to be seen whether other members of the PKC family follow the same activation scheme as suggested for PKCbII, but it is quite possible that other PKCs and AGC kinases take advantage of the flexibility of the NFD motif with similar autoinhibitory clamps. Additional regulatory mechanisms involving specific protein interactions or posttranslational modification may also exert their effects through the NFD motif.
REFERENCES Canagarajah, B., Leskow, F.C., Ho, J.Y., Mischak, H., Saidi, L.F., Kazanietz, M.G., and Hurley, J.H. (2004). Cell 119, 407–418. Colon-Gonzalez, F., and Kazanietz, M.G. (2006). Biochim. Biophys. Acta 1761, 827–837. Colon-Gonzalez, F., Coluccio Leskow, F., and Kazanietz, M.G. (2008). J. Biol. Chem. 283, 35247–35257. Grodsky, N., Li, Y., Bouzida, D., Love, R., Jensen, J., Nodes, B., Nonomiya, J., and Grant, S. (2006). Biochemistry 45, 13970–13981. Kannan, N., Haste, N., Taylor, S.S., and Neuwald, A.F. (2007). Proc. Natl. Acad. Sci. USA 104, 1272–1277. _ Leonard, T.A., Ro´zycki, B., Saidi, L.F., Hummer, G., and Hurley, J.H. (2011). Cell 144, 55–66. Newton, A.C. (2009). J. Lipid Res. 50, S266–S271. Pearce, L.R., Komander, D., and Alessi, D.R. (2010). Nat. Rev. Mol. Cell Biol. 11, 9–22. Stebbins, E.G., and Mochly-Rosen, D. (2001). J. Biol. Chem. 276, 29644–29650. Takimura, T., Kamata, K., Fukasawa, K., Ohsawa, H., Komatani, H., Yoshizumi, T., Takahashi, I., Kotani, H., and Iwasawa, Y. (2010). Acta Crystallogr. D Biol. Crystallogr. 66, 577–583.
Previews Garneau, J.E., Dupuis, M.E., Villion, M., Romero, D.A., Barrangou, R., Boyaval, P., Fremaux, C., Horvath, P., Magadan, A.H., and Moineau, S. (2010). Nature 468, 67–71. Haft, D., Selengut, J., Mongodin, E., and Nelson, K. (2005). PLoS Comput. Biol. 1, e60. Hale, C.R., Zhao, P., Olson, S., Duff, M.O., Graveley, B.R., Wells, L., Terns, R.M., and Terns, M.P. (2009). Cell 139, 945–956.
Haurwitz, R.E., Jinek, M., Wiedenheft, B., Zhou, K., and Doudna, J.A. (2010). Science 329, 1355–1358.
Marraffini, L.A., and Sontheimer, E.J. (2008). Science 322, 1843–1845.
Horvath, P., and Barrangou, R. (2010). Science 327, 167–170.
Mojica, F.J., Diez-Villasenor, C., Soria, E., and Juez, G. (2000). Mol. Microbiol. 36, 244–246.
Kunin, V., Sorek, R., and Hugenholtz, P. (2007). Genome Biol. 8, R61. Makarova, K.S., Grishin, N.V., Shabalina, S.A., Wolf, Y.I., and Koonin, E.V. (2006). Biol. Direct 1, 7.
Sorek, R., Kunin, V., and Hugenholtz, P. (2008). Nat. Rev. Microbiol. 6, 181–186. Wang, R., Preamplume, G., Terns, M.P., Terns, R.M., and Li, H. (2011). Structure 19, this issue, 257–264.
Protein Kinase C Regulation: C1 Meets C-tail Marcelo G. Kazanietz1,* and Mark A. Lemmon2,* 1Department
of Pharmacology of Biochemistry and Biophysics University of Pennsylvania School of Medicine, Philadelphia, PA, USA *Correspondence: [email protected] (M.G.K.), [email protected] (M.A.L.) DOI 10.1016/j.str.2011.01.004 2Department
In a recent issue of Cell, Leonard et al. (2011) describe the structure of PKCbII, an AGC kinase, revealing an unanticipated intramolecular autoinhibitory interaction between its C-terminal tail and the diacylglycerol and phorbol ester binding site of its C1b domain.
The three-dimensional structure of an intact protein kinase C (PKC), an archetypal lipid membrane effector, has long been anticipated. Following determination of the kinase domain structures for several AGC family Ser-Thr kinases (named after prominent members of the families PKA, PKG, and PKC), key questions have focused on how the kinase domain is regulated by other domains in these tightly controlled modular proteins. In the January 7 issue of Cell, Leonard et al. (2011) describe an important step toward this goal, with a 4 A˚ resolution structure of PKCbII that contains almost 80% of the intact protein–lacking only one of the regulatory domains. The new structure, supported by biochemical and small-angle X-ray scattering studies, adds an important new twist to our understanding of the multistep regulation of PKC. PKC isozymes are widely implicated in human physiology and disease, and their involvement in cancer progression is well established. PKCs are described as classical (cPKCa, bI, bII, and g), novel (nPKCd, 3, h, and q), or atypical (aPKCz and l/i) based on their biochemical requirements
for activation. Only cPKCs respond to calcium, whereas both cPKCs and nPKCs are sensitive to phorbol esters and the lipid second messenger diacylglycerol (DAG). Activation of PKCs is well known to proceed through multiple steps, the first of which involves transphosphorylation (required for maturation to an activatable state) and autophosphorylation. Even once ‘‘primed’’ by phosphorylation, PKC isoforms remain ‘‘autoinhibited’’ by intramolecular interactions between the N-terminal pseudosubstrate region and the active site of the C-terminal kinase domain (Figure 1A). Relieving this autoinhibition also involves multiple steps. In cPKCs, the C2 domain first binds Ca2+ and phospholipids, targeting the enzyme to the membrane. Once membrane-associated, binding of the C1 domains to DAG is thought to reverse autoinhibition by the pseudosubstrate region, leading to full activation of the enzyme (Newton, 2009). Now, the work from Hurley’s group yields two major new findings. First, the structure reveals an additional step in the allosteric activation of PKCbII. The kinase domain appears to be maintained in an inactive state by unexpected intra-
144 Structure 19, February 9, 2011 ª2011 Elsevier Ltd All rights reserved
molecular autoinhibitory interactions between the regulatory C1b domain and an ‘‘NFD motif’’ in the C-terminal tail. Second, the work argues that the two C1 domains in PKCbII (C1a and C1b) play substantially different roles, as predicted from several biochemical and pharmacological studies (Colon-Gonzalez and Kazanietz, 2006) (see below). As with all AGC kinases, the C-terminal tail of PKCbII (residues 621-673) is an important cis-acting regulatory module, wrapping around the N-lobe of the kinase domain (Figure 1B) to stabilize its active conformation (Pearce et al., 2010). The hydrophobic motif in the C-tail (residues 656-661) helps position the key aC helix in the active structure, and the flexible ‘‘turn motif’’ (residues 624-650) contributes to the ATP binding site (Grodsky et al., 2006). In PKCbII, phosphorylation of these regulatory elements promotes their ability to stabilize the active kinase. Just as these C-tail interactions are crucial for stabilizing the active kinase, they also provide vulnerability that seems to be exploited in PKCbII regulation. The structure reported by Leonard et al. (2011) reveals an intriguing intramolecular
Structure
Previews A
C-tail
B
C1b N-lobe ATP
NFD helix
F629 C-lobe
C-tail
C-tail
C C1b
N ATP
N
F629
F629
-P
C
active
NFD helix
ATP
-P
C
autoinhibited
Figure 1. PKCbII Autoinhibition (A) Domain composition of PKCbII. PS designates the pseudosubstrate region. The C-terminal tail is colored red. (B) Structure of kinase domain and C1b domain regions of PKCbII (from PDB ID 3PFQ). Kinase domain is gray, C-tail is red, and C1b domain is cyan. F629 in the NFD motif is contained within a helix that limits its access to the ATP binding site. The NFD helix and adjacent sequences project into the C1b DAG binding site. (C) Cartoon representation of PKCbII autoinhibition. Without C1b domain interaction, the NFD helix is ‘‘unwound,’’ and F629 can contribute to the ATP binding site. The intramolecular interaction with the C1b domain stabilizes helix formation in the NFD motif, rotating F629 out of the ATP binding site, leading to inhibition of the kinase.
interaction in which the C1b domain of PKCbII docks onto a binding site comprising residues 619-633 from the regulatory C-tail plus conserved elements in the kinase domain N-lobe. Moreover, it is the DAG binding site of the C1b domain that mediates this intramolecular contact. Although intramolecular interactions between the C1 domains and the rest of PKCbII might have been expected, their
consequence for the conformation of the kinase domain was not. The region of the C-tail that interacts with the C1b domain is one that alternatively plays an important role in stabilizing the active site of the kinase domain, and has been termed the ‘‘active-site tether’’ (Kannan et al., 2007). In protein kinase A (PKA), the side chain of a conserved phenylalanine (F327) in this region projects into
the ATP-binding pocket and contacts the bound nucleotide. In the PKCbII structure, the corresponding phenylalanine (F629) instead contacts the C1b domain and is prevented from participating in ATP binding (Figure 1B). This key phenylalanine lies in what Hurley and coworkers call the ‘‘NFD motif.’’ The NFD motif is in a region previously noted to be flexible in PKCbII, and forms an a helix both in the new PKCbII structure and in a structure of the isolated inhibitor-bound PKCbII kinase domain (Grodsky et al., 2006). By contrast, this helix is not seen in an ATPbound active PKC kinase domain structure (Takimura et al., 2010), where the NFD motif phenylalanine is freed to project into the ATP-binding site (Figure 1C). These structural details suggest an attractive model. In the absence of DAG, PKCbII is subject to at least two modes of autoinhibition. One involves the pseudosubstrate region. The second arises because the DAG-binding site of the C1b domain associates with the NFD motif in the regulatory C-tail, and ‘‘clamps’’ it in a position that is suboptimal for kinase activity (Figure 1C). In doing so, an a helix is formed in the NFD region, which appears to be characteristic of the inactive state. Only when the C1b domain is engaged by DAG (or phorbol esters) is this clamp released (and the NFD helix unfolded), allowing the kinase to adopt its fully active configuration. Other autoinhibitory sites may also need to be released, such as those involving isozyme-specific motifs required for intracellular compartmentalization (Stebbins and Mochly-Rosen, 2001).
The C1 Domain: More than a Lipid-Binding Motif DAG or phorbol ester binding to C1 domains confers a high-affinity interaction between PKC and the membrane. Tandem C1 domains are found in cPKCs, nPKCs, and other kinases (protein kinase D isozymes and DAG kinases). Other signaling proteins have only one C1 domain, including MRCK (myotonic dystrophy kinase-related Cdc42 binding kinase), Munc-13 scaffolding proteins, and small G protein regulators such as RasGRPs (guanine nucleotide exchangers) or the a- and b-chimaerins (Rac GTPase activating proteins, or RacGAPs).
Structure 19, February 9, 2011 ª2011 Elsevier Ltd All rights reserved 145
Structure
Previews As mentioned above, a major suggestion of the Hurley paper is that the C1a and C1b domains in PKCbII have distinct roles. Indeed, the functional nonequivalence of C1a and C1b became evident in early analyses of membrane translocation of C1 domain mutants and later from biochemical studies (Colon-Gonzalez and Kazanietz, 2006). Despite significant sequence homology, individual C1 domains also differ in their ability to bind ligands and penetrate membranes (Colon-Gonzalez and Kazanietz, 2006). According to the model postulated by Hurley and coworkers, the C1a domain of PKCbII is exposed and is the first to bind DAG in the membrane after initial priming of PKC by calcium. This step may provide the energy for disrupting autoinhibitory interactions between the N-terminal pseudosubstrate region and the kinase domain. Full kinase activation then occurs only after the C1b clamp is released from the NFD motif, coincident with its association with DAG. Despite the obvious differences between PKCs and proteins that contain a single C1 domain, there are similarities between the mechanisms of regulation of PKCbII and b2-chimaerin, for example. In both cases, the ‘‘closed’’ conformation is stabilized by C1 domain association with other domains in the same protein, with which DAG/phorbol ester and phospholipid binding must compete. Like the C1b domain in the Hurley PKCbII structure, the C1 domain in b2-chimaerin is buried, contacting the catalytic (Rac-GAP) domain and other regions. Disrupting the intramolecular C1 domain
interactions by mutagenesis renders b2-chimaerin more sensitive to phorbol 12-myristate 13-acetate (PMA)-induced translocation to membranes and confers elevated Rac-GAP activity (Canagarajah et al., 2004). Leonard et al. (2011) see a similar effect when they disrupt the crystallographically observed clamp in PKCbII. Interestingly, the chimaerins require a higher concentration of PMA for translocation than PKCs do, possibly reflecting a more extensive C1 domain burial. However, in response to physiological stimuli such as EGF, which generates DAG through activation of phospholipase C, full translocation of chimaerins is observed despite the lower affinity of C1 domains for DAG than for PMA (Colon-Gonzalez et al., 2008). DAG binding to the C1 domain is required, but not sufficient, for chimaerin translocation, suggesting that other factors contribute to disruption of the intramolecular autoinhibitory interactions. One possibility is that association of other proteins with the chimaerin N-terminal region weakens intramolecular C1 domain interactions. Interaction of PKCs with other proteins appears crucial for dictating the distinctive intracellular localization of each isoform, and thus their differential access to substrates. The C1 domains may play a role in this. Indeed, both C1a and C1b domains have been shown to engage in protein-protein interactions, one key example being the association of p23/Tmp21 with the C1b domain of nPKCs, anchoring them in the perinuclear region (Colon-Gonzalez and Kazanietz, 2006).
146 Structure 19, February 9, 2011 ª2011 Elsevier Ltd All rights reserved
It remains to be seen whether other members of the PKC family follow the same activation scheme as suggested for PKCbII, but it is quite possible that other PKCs and AGC kinases take advantage of the flexibility of the NFD motif with similar autoinhibitory clamps. Additional regulatory mechanisms involving specific protein interactions or posttranslational modification may also exert their effects through the NFD motif.
REFERENCES Canagarajah, B., Leskow, F.C., Ho, J.Y., Mischak, H., Saidi, L.F., Kazanietz, M.G., and Hurley, J.H. (2004). Cell 119, 407–418. Colon-Gonzalez, F., and Kazanietz, M.G. (2006). Biochim. Biophys. Acta 1761, 827–837. Colon-Gonzalez, F., Coluccio Leskow, F., and Kazanietz, M.G. (2008). J. Biol. Chem. 283, 35247–35257. Grodsky, N., Li, Y., Bouzida, D., Love, R., Jensen, J., Nodes, B., Nonomiya, J., and Grant, S. (2006). Biochemistry 45, 13970–13981. Kannan, N., Haste, N., Taylor, S.S., and Neuwald, A.F. (2007). Proc. Natl. Acad. Sci. USA 104, 1272–1277. _ Leonard, T.A., Ro´zycki, B., Saidi, L.F., Hummer, G., and Hurley, J.H. (2011). Cell 144, 55–66. Newton, A.C. (2009). J. Lipid Res. 50, S266–S271. Pearce, L.R., Komander, D., and Alessi, D.R. (2010). Nat. Rev. Mol. Cell Biol. 11, 9–22. Stebbins, E.G., and Mochly-Rosen, D. (2001). J. Biol. Chem. 276, 29644–29650. Takimura, T., Kamata, K., Fukasawa, K., Ohsawa, H., Komatani, H., Yoshizumi, T., Takahashi, I., Kotani, H., and Iwasawa, Y. (2010). Acta Crystallogr. D Biol. Crystallogr. 66, 577–583.