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Protein Kinase C Regulation: C1 Meets C-tail

2011; Elsevier BV; Volume: 19; Issue: 2 Linguagem: Inglês

10.1016/j.str.2011.01.004

1878-4186

Marcelo G. Kazanietz, Mark A. Lemmon,

Coagulation, Bradykinin, Polyphosphates, and Angioedema

In a recent issue of Cell, Leonard et al., 2011Leonard T.A. Różycki B. Saidi L.F. Hummer G. Hurley J.H. Cell. 2011; 144: 55-66Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar describe the structure of PKCβII, 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. In a recent issue of Cell, Leonard et al., 2011Leonard T.A. Różycki B. Saidi L.F. Hummer G. Hurley J.H. Cell. 2011; 144: 55-66Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar describe the structure of PKCβII, 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., 2011Leonard T.A. Różycki B. Saidi L.F. Hummer G. Hurley J.H. Cell. 2011; 144: 55-66Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar describe an important step toward this goal, with a 4 Å resolution structure of PKCβII 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 (cPKCα, βI, βII, and γ), novel (nPKCδ, ɛ, η, and θ), or atypical (aPKCζ and λ/ι) 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, 2009Newton A.C. J. Lipid Res. 2009; 50: S266-S271Crossref PubMed Scopus (120) Google Scholar). Now, the work from Hurley's group yields two major new findings. First, the structure reveals an additional step in the allosteric activation of PKCβII. The kinase domain appears to be maintained in an inactive state by unexpected intramolecular 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 PKCβII (C1a and C1b) play substantially different roles, as predicted from several biochemical and pharmacological studies (Colon-Gonzalez and Kazanietz, 2006Colon-Gonzalez F. Kazanietz M.G. Biochim. Biophys. Acta. 2006; 1761: 827-837Crossref PubMed Scopus (210) Google Scholar) (see below). As with all AGC kinases, the C-terminal tail of PKCβII (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., 2010Pearce L.R. Komander D. Alessi D.R. Nat. Rev. Mol. Cell Biol. 2010; 11: 9-22Crossref PubMed Scopus (891) Google Scholar). The hydrophobic motif in the C-tail (residues 656-661) helps position the key αC helix in the active structure, and the flexible “turn motif” (residues 624-650) contributes to the ATP binding site (Grodsky et al., 2006Grodsky N. Li Y. Bouzida D. Love R. Jensen J. Nodes B. Nonomiya J. Grant S. Biochemistry. 2006; 45: 13970-13981Crossref PubMed Scopus (87) Google Scholar). In PKCβII, 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 PKCβII regulation. The structure reported by Leonard et al., 2011Leonard T.A. Różycki B. Saidi L.F. Hummer G. Hurley J.H. Cell. 2011; 144: 55-66Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar reveals an intriguing intramolecular interaction in which the C1b domain of PKCβII 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 PKCβII 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., 2007Kannan N. Haste N. Taylor S.S. Neuwald A.F. Proc. Natl. Acad. Sci. USA. 2007; 104: 1272-1277Crossref PubMed Scopus (162) Google Scholar). 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 PKCβII 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 PKCβII, and forms an α helix both in the new PKCβII structure and in a structure of the isolated inhibitor-bound PKCβII kinase domain (Grodsky et al., 2006Grodsky N. Li Y. Bouzida D. Love R. Jensen J. Nodes B. Nonomiya J. Grant S. Biochemistry. 2006; 45: 13970-13981Crossref PubMed Scopus (87) Google Scholar). By contrast, this helix is not seen in an ATP-bound active PKC kinase domain structure (Takimura et al., 2010Takimura T. Kamata K. Fukasawa K. Ohsawa H. Komatani H. Yoshizumi T. Takahashi I. Kotani H. Iwasawa Y. Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 577-583Crossref PubMed Scopus (30) Google Scholar), 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, PKCβII 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 α 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, 2001Stebbins E.G. Mochly-Rosen D. J. Biol. Chem. 2001; 276: 29644-29650Crossref PubMed Scopus (143) Google Scholar). 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 α- and β-chimaerins (Rac GTPase activating proteins, or Rac-GAPs). As mentioned above, a major suggestion of the Hurley paper is that the C1a and C1b domains in PKCβII 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, 2006Colon-Gonzalez F. Kazanietz M.G. Biochim. Biophys. Acta. 2006; 1761: 827-837Crossref PubMed Scopus (210) Google Scholar). Despite significant sequence homology, individual C1 domains also differ in their ability to bind ligands and penetrate membranes (Colon-Gonzalez and Kazanietz, 2006Colon-Gonzalez F. Kazanietz M.G. Biochim. Biophys. Acta. 2006; 1761: 827-837Crossref PubMed Scopus (210) Google Scholar). According to the model postulated by Hurley and coworkers, the C1a domain of PKCβII 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 PKCβII and β2-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 PKCβII structure, the C1 domain in β2-chimaerin is buried, contacting the catalytic (Rac-GAP) domain and other regions. Disrupting the intramolecular C1 domain interactions by mutagenesis renders β2-chimaerin more sensitive to phorbol 12-myristate 13-acetate (PMA)-induced translocation to membranes and confers elevated Rac-GAP activity (Canagarajah et al., 2004Canagarajah B. Leskow F.C. Ho J.Y. Mischak H. Saidi L.F. Kazanietz M.G. Hurley J.H. Cell. 2004; 119: 407-418Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Leonard et al., 2011Leonard T.A. Różycki B. Saidi L.F. Hummer G. Hurley J.H. Cell. 2011; 144: 55-66Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar see a similar effect when they disrupt the crystallographically observed clamp in PKCβII. 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., 2008Colon-Gonzalez F. Coluccio Leskow F. Kazanietz M.G. J. Biol. Chem. 2008; 283: 35247-35257Crossref PubMed Scopus (25) Google Scholar). 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, 2006Colon-Gonzalez F. Kazanietz M.G. Biochim. Biophys. Acta. 2006; 1761: 827-837Crossref PubMed Scopus (210) Google Scholar). It remains to be seen whether other members of the PKC family follow the same activation scheme as suggested for PKCβII, 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. Crystal Structure and Allosteric Activation of Protein Kinase C βIILeonard et al.CellJanuary 07, 2011In BriefProtein kinase C (PKC) isozymes are the paradigmatic effectors of lipid signaling. PKCs translocate to cell membranes and are allosterically activated upon binding of the lipid diacylglycerol to their C1A and C1B domains. The crystal structure of full-length protein kinase C βII was determined at 4.0 Å, revealing the conformation of an unexpected intermediate in the activation pathway. Here, the kinase active site is accessible to substrate, yet the conformation of the active site corresponds to a low-activity state because the ATP-binding side chain of Phe629 of the conserved NFD motif is displaced. Full-Text PDF Open Archive