Protein kinase C-δ (PKCδ) is a signalling kinase that regulates many cellular responses. Although most studies focus on allosteric mechanisms that activate PKCδ at membranes, PKCδ also is controlled via multi-site phosphorylation [Gong et al. (2015) Mol. Cell. Biol. 35, 1727–1740]. The present study uses MS-based methods to identify PKCδ phosphorylation at Thr50 and Ser645 (in resting and PMA-treated cardiomyocytes) as well as Thr37, Thr38, Ser130, Thr164, Thr211, Thr215, Ser218, Thr295, Ser299 and Thr656 (as sites that increase with PMA). We focused on the consequences of phosphorylation at Ser130 and Thr141 (sites just N-terminal to the pseudosubstrate domain). We show that S130D and T141E substitutions co-operate to increase PKCδ’s basal lipid-independent activity and that Ser130/Thr141 di-phosphorylation influences PKCδ’s substrate specificity. We recently reported that PKCδ preferentially phosphorylates substrates with a phosphoacceptor serine residue and that this is due to constitutive phosphorylation at Ser357, an ATP-positioning G-loop site that limits PKCδ’s threonine kinase activity [Gong et al. (2015) Mol. Cell. Biol. 35, 1727–1740]. The present study shows that S130D and T141E substitutions increase PKCδ’s threonine kinase activity indirectly by decreasing G loop phosphorylation at Ser357. A S130F substitution [that mimics a S130F single-nt polymorphism (SNP) identified in some human populations] also increases PKCδ’s maximal lipid-dependent catalytic activity and confers threonine kinase activity. Finally, we show that Ser130/Thr141 phosphorylations relieve auto-inhibitory constraints that limit PKCδ’s activity and substrate specificity in a cell-based context. Since phosphorylation sites map to similar positions relative to the pseudosubstrate domains of other PKCs, our results suggest that phosphorylation in this region of the enzyme may constitute a general mechanism to control PKC isoform activity.
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February 2016
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Point scanning confocal microscopic imaging of C2C12 undifferentiatedmyoblasts (top panels) and differentiatedmultinucleated myotubes (bottom panels) immunofluorescently labelled with DAPI (blue) PDLIM7 (green) and Phalloidin or Nedd4-1 (red). Merged images show co-localization of PDLIM7 and Phalloidin decreases with myotube formation (left panels) and co-location of PDLIM7 and Nedd4-1 increases with myotube formation (right panels). Image courtesy of Robert D’Cruz et al. (for further details see pages 267–276). - PDF Icon PDF LinkFront Matter
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Research Article|
January 25 2016
A novel phosphorylation site at Ser130 adjacent to the pseudosubstrate domain contributes to the activation of protein kinase C-δ
Jianli Gong;
Jianli Gong
*Department of Pharmacology, Columbia University, New York, NY 10032, U.S.A.
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Ronald J. Holewinski;
Ronald J. Holewinski
†Advanced Clinical Biosystems Research Institute, The Heart Institute, Cedars Sinai Medical Center, Los Angeles, CA 90048, U.S.A.
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Jennifer E. Van Eyk;
Jennifer E. Van Eyk
†Advanced Clinical Biosystems Research Institute, The Heart Institute, Cedars Sinai Medical Center, Los Angeles, CA 90048, U.S.A.
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Susan F. Steinberg
Susan F. Steinberg
1
*Department of Pharmacology, Columbia University, New York, NY 10032, U.S.A.
1To whom correspondence should be addressed (email sfs1@columbia.edu).
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Publisher: Portland Press Ltd
Received:
July 21 2015
Revision Received:
October 19 2015
Accepted:
November 06 2015
Accepted Manuscript online:
November 06 2015
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 2016 Authors; published by Portland Press Limited
2016
Biochem J (2016) 473 (3): 311–320.
Article history
Received:
July 21 2015
Revision Received:
October 19 2015
Accepted:
November 06 2015
Accepted Manuscript online:
November 06 2015
Citation
Jianli Gong, Ronald J. Holewinski, Jennifer E. Van Eyk, Susan F. Steinberg; A novel phosphorylation site at Ser130 adjacent to the pseudosubstrate domain contributes to the activation of protein kinase C-δ. Biochem J 1 February 2016; 473 (3): 311–320. doi: https://doi.org/10.1042/BJ20150812
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