Dataset Information

The C2 Domain and Altered ATP-Binding Loop Phosphorylation at Ser³?? Mediate the Redox-Dependent Increase in Protein Kinase C-? Activity.

ABSTRACT: The diverse roles of protein kinase C-? (PKC?) in cellular growth, survival, and injury have been attributed to stimulus-specific differences in PKC? signaling responses. PKC? exerts membrane-delimited actions in cells activated by agonists that stimulate phosphoinositide hydrolysis. PKC? is released from membranes as a Tyr(313)-phosphorylated enzyme that displays a high level of lipid-independent activity and altered substrate specificity during oxidative stress. This study identifies an interaction between PKC?'s Tyr(313)-phosphorylated hinge region and its phosphotyrosine-binding C2 domain that controls PKC?'s enzymology indirectly by decreasing phosphorylation in the kinase domain ATP-positioning loop at Ser(359). We show that wild-type (WT) PKC? displays a strong preference for substrates with serine as the phosphoacceptor residue at the active site when it harbors phosphomimetic or bulky substitutions at Ser(359.) In contrast, PKC?-S359A displays lipid-independent activity toward substrates with either a serine or threonine as the phosphoacceptor residue. Additional studies in cardiomyocytes show that oxidative stress decreases Ser(359) phosphorylation on native PKC? and that PKC?-S359A overexpression increases basal levels of phosphorylation on substrates with both phosphoacceptor site serine and threonine residues. Collectively, these studies identify a C2 domain-pTyr(313) docking interaction that controls ATP-positioning loop phosphorylation as a novel, dynamically regulated, and physiologically relevant structural determinant of PKC? catalytic activity.

SUBMITTER: Gong J

PROVIDER: S-EPMC4405634 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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Publications

The C2 Domain and Altered ATP-Binding Loop Phosphorylation at Ser³⁵⁹ Mediate the Redox-Dependent Increase in Protein Kinase C-δ Activity.

Gong Jianli J Yao Yongneng Y Zhang Pingbo P Udayasuryan Barath B Komissarova Elena V EV Chen Ju J Sivaramakrishnan Sivaraj S Van Eyk Jennifer E JE Steinberg Susan F SF

Molecular and cellular biology 20150309 10

The diverse roles of protein kinase C-δ (PKCδ) in cellular growth, survival, and injury have been attributed to stimulus-specific differences in PKCδ signaling responses. PKCδ exerts membrane-delimited actions in cells activated by agonists that stimulate phosphoinositide hydrolysis. PKCδ is released from membranes as a Tyr(313)-phosphorylated enzyme that displays a high level of lipid-independent activity and altered substrate specificity during oxidative stress. This study identifies an intera ...[more]

PMID: 25755284

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Project description:Chk2 is a critical regulator of the cellular DNA damage repair response. Activation of Chk2 in response to IR-induced damage is initiated by phosphorylation of the Chk2 SQ/TQ cluster domain at Ser(19), Ser(33), Ser(35), and Thr(68). This precedes autophosphorylation of Thr(383)/Thr(387) in the T-loop region of the kinase domain an event that is a prerequisite for efficient kinase activity. We conducted an in-depth analysis of phosphorylation within the T-loop region (residues 366-406). We report four novel phosphorylation sites at Ser(372), Thr(378), Thr(389), and Tyr(390). Substitution mutation Y390F was defective for kinase function. The substitution mutation T378A ablated the IR induction of kinase activity. Interestingly, the substitution mutation T389A demonstrated a 6-fold increase in kinase activity when compared with wild-type Chk2. In addition, phosphorylation at Thr(389) was a prerequisite to phosphorylation at Thr(387) but not at Thr(383). Quantitative mass spectrometry analysis revealed IR-induced phosphorylation and subcellular distribution of Chk2 phosphorylated species. We observed IR-induced increase in phosphorylation at Ser(379), Thr(389), and Thr(383)/Thr(389). Phosphorylation at Tyr(390) was dramatically reduced following IR. Exposure to IR was also associated with changes in the ratio of chromatin/nuclear localization. IR-induced increase in chromatin localization was associated with phosphorylation at Thr(372), Thr(379), Thr(383), Thr(389), Thr(383)/Thr(387), and Thr(383)/Thr(389). Chk2 hyper-phosphorylated species at Thr(383)/Thr(387)/Thr(389) and Thr(383)/Thr(387)/Thr(389)/Tyr(390) relocalized from almost exclusively chromatin to predominately nuclear expression, suggesting a role for phosphorylation in regulation of chromatin targeting and egress. The differential impact of T-loop phosphorylation on Chk2 ubiquitylation suggests a co-dependence of these modifications. The results demonstrate that a complex interdependent network of phosphorylation events within the T-loop exchange region regulates dimerization/autophosphorylation, kinase activation, and chromatin targeting/egress of Chk2.

Dataset Information

The C2 Domain and Altered ATP-Binding Loop Phosphorylation at Ser³?? Mediate the Redox-Dependent Increase in Protein Kinase C-? Activity.

Publications

The C2 Domain and Altered ATP-Binding Loop Phosphorylation at Ser³⁵⁹ Mediate the Redox-Dependent Increase in Protein Kinase C-δ Activity.

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