Independence of protein kinase C-delta activity from activation loop phosphorylation - Structural basis and altered functions in cells

Experimental Immunology Branch, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Journal of Biological Chemistry (Impact Factor: 4.6). 05/2006; 281(17):12102-11. DOI: 10.1074/jbc.M600508200
Source: PubMed

ABSTRACT Activation loop phosphorylation plays critical regulatory roles for many kinases. Unlike other protein kinase Cs (PKC), PKC-delta does not require phosphorylation of its activation loop (Thr-507) for in vitro activity. We investigated the structural basis for this unusual capacity and its relevance to PKC-delta function in intact cells. Mutational analysis demonstrated that activity without Thr-507 phosphorylation depends on 20 residues N-terminal to the kinase domain and a pair of phenylalanines (Phe-500/Phe-527) unique to PKC-delta in/near the activation loop. Molecular modeling demonstrated that these elements stabilize the activation loop by forming a hydrophobic chain of interactions from the C-lobe to activation loop to N-terminal (helical) extension. In cells PKC-delta mediates both apoptosis and transcription regulation. We found that the T507A mutant of the PKC-delta kinase domain resembled the corresponding wild type in mediating apoptosis in transfected HEK293T cells. But the T507A mutant was completely defective in AP-1 and NF-kappaB reporter assays. A novel assay in which the kinase domain of PKC-delta and its substrate (a fusion protein of PKC substrate peptide with green fluorescent protein) were co-targeted to lipid rafts revealed a major substrate-selective defect of the T507A mutant in phosphorylating the substrate in cells. In vitro analysis showed strong product inhibition on the T507A mutant with particular substrates whose characteristics suggest it contributes to the substrate selective defect of the PKC-delta T507A mutant in cells. Thus, activation loop phosphorylation of PKC-delta may regulate its function in cells in a novel way.

  • [Show abstract] [Hide abstract]
    ABSTRACT: The objective of this study was to elucidate the signaling pathway through which cationic antimicrobial protein of 37 kDa (CAP37) mediates human corneal epithelial cell (HCEC) chemotaxis. Immortalized human corneal epithelial cells (HCEC) were treated with pertussis toxin (10 and 1000 ng/ml), protein kinase C (PKC) inhibitors (calphostin c, 50 nM and Ro 31-8220, 100 nM), phorbol esters (phorbol 12,13-dibutyrate, 200 nM and phorbol 12-myristate 13-acetate, 1 μM) known to deplete PKC isoforms, and siRNAs (400 nM) before a modified Boyden chamber assay was used to determine the effect of these inhibitors and siRNAs on CAP37-directed HCEC migration. PKCδ protein levels, PKCδ-Thr(505)phosphorylation, and PKCδ kinase activity was assessed in CAP37 treated HCECs using immunohistochemistry, Western blotting, and a kinase activity assay respectively. Chemotaxis studies revealed that treatment with pertussis toxin, PKC inhibitors, phorbol esters, and siRNAs significantly inhibited CAP37-mediated chemotaxis compared to untreated controls. CAP37 treatment increased PKCδ protein levels and led to PKCδ phosphorylation on residue Thr505. Direct activation of PKCδ by CAP37 was demonstrated using a kinase activity assay. These findings lead us to conclude that CAP37 is an important regulator of corneal epithelial cell migration and mediates its effects through PKCδ
    Investigative ophthalmology & visual science 09/2013; 54(10). DOI:10.1167/iovs.13-12054 · 3.66 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Fission yeast PKC ortholog Pck2 controls cell wall synthesis and is a major upstream activator of the cell integrity pathway (CIP) and its core component, MAP kinase Pmk1, in response to environmental stimuli. We show that in vivo phosphorylation of Pck2 at the conserved T842 activation loop during growth and in response to different stresses is mediated by the PDK ortholog Ksg1 and an autophosphorylation mechanism. However, T842 phosphorylation is not essential for Pmk1 activation, and putative phosphorylation at T846 might play an additional role for Pck2 catalytic activation and downstream signaling. These events together with turn motif autophosphorylation at T984 and binding to small GTPases Rho1 and/or Rho2 stabilize and render Pck2 competent to exert its biological functions. Remarkably, the TORC2 complex does not participate in catalytic activation of Pck2, but instead contributes to de novo Pck2 synthesis which is essential to activate the CIP in response to cell wall damage or glucose exhaustion. These results unveil a novel mechanism whereby TOR regulates PKC function at a translational level and add a new regulatory layer to MAPK signaling cascades.
    Journal of Cell Science 11/2014; 128(2). DOI:10.1242/jcs.158295 · 5.33 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The wood frog, Rana sylvatica, survives whole-body freezing and thawing each win-ter. The extensive adaptations required at the biochemical level are facilitated by alterations to signaling pathways, including the insulin/Akt and AMPK pathways. Past studies investigating changing tissue-specific patterns of the second messenger IP 3 in adapted frogs have suggested important roles for protein kinase C (PKC) in response to stress. In addition to their dependence on second messengers, phos-phorylation of three PKC sites by upstream kinases (most notably PDK1) is needed for full PKC activation, according to widely-accepted models. The present study uses phospho-specific immunoblotting to investigate phosphorylation states of PKC—as they relate to distinct tissues, PKC isozymes, and phosphorylation sites— in control and frozen frogs. In contrast to past studies where second messengers of PKC increased during the freezing process, phosphorylation of PKC tended to generally decline in most tissues of frozen frogs. All PKC isozymes and specific phosphorylation sites detected by immunoblotting decreased in phosphorylation levels in hind leg skeletal muscle and hearts of frozen frogs. Most PKC isozymes and specific phosphorylation sites detected in livers and kidneys also declined; the only exceptions were the levels of isozymes/phosphorylation sites detected by the phospho-PKCα/βII (Thr638/641) antibody, which remained unchanged from con-trol to frozen frogs. Changes in brains of frozen frogs were unique; no decreases were observed in the phosphorylation levels of any of the PKC isozymes and/or specific phosphorylation sites detected by immunoblotting. Rather, increases were observed for the levels of isozymes/phosphorylation sites detected by the phospho-PKCα/βII (Thr638/641), phospho-PKCδ (Thr505), and phospho-PKCθ (Thr538) antibodies; all other isozymes/phosphorylation sites detected in brain remained unchanged from control to frozen frogs. The results of this study indicate a potential important role for PKC in cerebral protection during wood frog freezing. Our findings also call for a reassessment of the previously-inferred importance of PKC in other tissues, particularly in liver; a more thorough investigation is required to determine whether PKC activity in this physiological situation is indeed dependent on phosphorylation, or whether it deviates from the generally-accepted model and can be "overridden" by exceedingly high levels of second messengers, as has been demonstrated with certain PKC isozymes (e.g., PKCδ). How to cite this article Dieni and Storey (2014), Protein kinase C in the wood frog, Rana sylvatica: reassessing the tissue-specific regulation of PKC isozymes during freezing. PeerJ 2:e558; DOI 10.7717/peerj.558
    09/2004; 2:e558. DOI:10.7717/peerj.558