The Last 10 Amino Acid Residues beyond the Hydrophobic Motif Are Critical for the Catalytic Competence and Function of Protein Kinase Cα

Yeong, S.S.; Zhu, Yimin; Smith, D. J.; Verma, Chandra; Lim, Wee Guan; Tan, Bee Jen; Li, Qiu Tian; Cheung, Nam Sang; Cai, Minnie; Zhu, Yi Zhun; Zhou, Shu Feng; Tan, Seng Lai; Duan, Wei

doi:10.1074/jbc.m511278200

Cited by 18 publications

(13 citation statements)

References 70 publications

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“…However, the PKC␣-⌬C-tail-mCit-FLAG sensor used in Fig. 6, a and c, lacks the priming phosphorylations and, as previously reported (31), has no detectable ATPase activity. To assess if this sensor retains any of its native tertiary structure in the catalytic domain and as such can provide useful information on the binding interface, we adopted an assay that has commonly been used to measure nucleotide binding in other ATPases, including in protein kinase A (32), but has to our knowledge not been tested with PKCs.…”

Section: Pkc␣ Homodimerizes Upon Stimulation With Effectors Insupporting

confidence: 69%

Conserved Modular Domains Team up to Latch-open Active Protein Kinase Cα

Swanson

Ritt²,

Wang³

et al. 2014

Journal of Biological Chemistry

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Background: Protein kinase C (PKC) is a nodal regulator of cell signaling. Results: Multiple interactions between conserved regulatory domains in PKC synergistically stabilize a nanomolar affinity homodimer critical for cellular function. Conclusion: Homodimerization regulates the equilibrium between the auto-inhibited and active states of PKC␣. Significance: Multiplexed interactions between modular domains dictate PKC function.

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Section: Pkc␣ Homodimerizes Upon Stimulation With Effectors Insupporting

confidence: 69%

Conserved Modular Domains Team up to Latch-open Active Protein Kinase Cα

Swanson

Ritt²,

Wang³

et al. 2014

Journal of Biological Chemistry

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“…If the C-terminal ten amino acids are removed from PKC␣ its catalytic activity is lost (32). This may reflect the fact that the V5 domain, when the autophosphorylation sites are phosphorylated, positions itself on the top of the N-lobe of the catalytic domain (33).…”

Section: Discussionmentioning

confidence: 99%

Identification of Acidic Amino Acid Residues in the Protein Kinase Cα V5 Domain That Contribute to Its Insensitivity to Diacylglycerol

Stensman

Larsson

2007

Journal of Biological Chemistry

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The protein kinase C (PKC) isoforms are maintained in an inactive and closed conformation by intramolecular interactions. Upon activation these are disrupted by activators, binding proteins and cellular membrane. We have seen that autophosphorylation of two sites in the C-terminal V5 domain is crucial to keep PKC␣ insensitive to the activator diacylglycerol, which presumably is caused by a masking of the diacylglycerol-binding C1a domain. Here we demonstrate that the diacylglycerol sensitivity of the PKC␤ isoforms also is suppressed by autophosphorylation of the V5 sites. To analyze conformational differences, a fusion protein ECFP-PKC␣-EYFP was expressed in cells and the FRET signal was analyzed. The analogous mutant with autophosphorylation sites exchanged for alanine gave rise to a substantially lower FRET signal than wild-type PKC␣ indicating a conformational difference elicited by the mutations. Expression of the isolated PKC␣ V5 domain led to increased diacylglycerol sensitivity of PKC␣. We identified acidic residues in the V5 domain that, when mutated to alanines or lysines, rendered PKC␣ sensitive to diacylglycerol. Furthermore, mutation to glutamate of four lysines in a lysine-rich cluster in the C2 domain gave a similar effect. Simultaneous reversal of the charges of the acidic residues in the V5 and the lysines in the C2 domain gave rise to a PKC␣ that was insensitive to diacylglycerol. We propose that these structures participate in an intramolecular interaction that maintains PKC␣ in a closed conformation. The disruption of this interaction leads to an unmasking of the C1a domain and thereby increased diacylglycerol sensitivity of PKC␣.The members of the protein kinase C (PKC) 2 family are important regulators of a multitude of cellular processes. The family consists of ten isoforms with distinct regulatory properties and functions. They are traditionally subgrouped in classical (PKC␣, ␤I, ␤II, and ␥), novel (PKC␦, ⑀, , and ) and atypical (PKC and ) PKC isoforms. The classification is based on the sensitivity of the isoforms to the PKC-activating second messengers Ca 2ϩ and 1,2-diacylglycerol. The classical isoforms respond to both activators whereas novel PKCs are insensitive to Ca 2ϩ but activated by diacylglycerol. The atypical isoforms are not regulated by any of the activators (1-3).In its inactive state, the PKC molecule is kept in a closed conformation by intramolecular interactions. This involves the binding of a pseudosubstrate motif (4), localized in the regulatory domain, to the substrate-binding site in the catalytic domain, thereby preventing the interaction of PKC with its substrates. To activate PKC second messengers interact with specific domains within the regulatory domain of the PKC molecule. Ca 2ϩ binds the C2 domain and diacylglycerol interacts with one or both of the C1 domains. This generally leads to a translocation of the enzyme to cellular membranes and a concomitant conformational change resulting in the release of the pseudosubstrate from the catalytic domain and thereby ...

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“…Yeong et al (233) recently published surprising evidence that the very distal portion of the V5 domain (COOH terminal to the hydrophobic motif phosphorylation site, that is absent in PKA) is critical for PKC α catalytic activity and function in cells (233). These investigators used a deletion approach to show that a single residue truncation at the extreme COOH terminus leads to a 60% reduction in enzyme activity (without any activation loop phosphorylation defect); truncation of the last 10 amino acids results in a PKC α mutant that lacks activation loop phosphorylation and is essentially catalytically inactive.…”

Section: The V5 Domainmentioning

confidence: 99%

Structural Basis of Protein Kinase C Isoform Function

Steinberg¹

2008

Physiological Reviews

773

827

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Protein kinase C (PKC) isoforms comprise a family of lipid-activated enzymes that have been implicated in a wide range of cellular functions. PKCs are modular enzymes comprised of a regulatory domain (that contains the membrane-targeting motifs that respond to lipid cofactors, and in the case of some PKCs calcium) and a relatively conserved catalytic domain that binds ATP and substrates. These enzymes are coexpressed and respond to similar stimulatory agonists in many cell types. However, there is growing evidence that individual PKC isoforms subserve unique (and in some cases opposing) functions in cells, at least in part as a result of isoform-specific subcellular compartmentalization patterns, protein-protein interactions, and posttranslational modifications that influence catalytic function. This review focuses on the structural basis for differences in lipid cofactor responsiveness for individual PKC isoforms, the regulatory phosphorylations that control the normal maturation, activation, signaling function, and downregulation of these enzymes, and the intra-/intermolecular interactions that control PKC isoform activation and subcellular targeting in cells. A detailed understanding of the unique molecular features that underlie isoform-specific posttranslational modification patterns, protein-protein interactions, and subcellular targeting (i.e., that impart functional specificity) should provide the basis for the design of novel PKC isoform-specific activator or inhibitor compounds that can achieve therapeutically useful changes in PKC signaling in cells.

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The Last 10 Amino Acid Residues beyond the Hydrophobic Motif Are Critical for the Catalytic Competence and Function of Protein Kinase Cα

Cited by 18 publications

References 70 publications

Conserved Modular Domains Team up to Latch-open Active Protein Kinase Cα

Conserved Modular Domains Team up to Latch-open Active Protein Kinase Cα

Identification of Acidic Amino Acid Residues in the Protein Kinase Cα V5 Domain That Contribute to Its Insensitivity to Diacylglycerol

Structural Basis of Protein Kinase C Isoform Function

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