Invariant Leu Preceding Turn Motif Phosphorylation Site Controls the Interaction of Protein Kinase C with Hsp70

Department of Pharmacology, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 11/2006; 281(43):32461-8. DOI: 10.1074/jbc.M604076200
Source: PubMed


Heat shock proteins play important roles in regulating signal transduction in cells by associating with, and stabilizing, diverse signaling molecules, including protein kinases. Previously, we have shown that heat shock protein Hsp70 associates with protein kinase C (PKC) via an interaction that is triggered by dephosphorylation at the turn phosphorylation motif. Here we have identified an invariant residue in the carboxyl terminus of PKC that mediates the binding to Hsp70. Specifically, we show that Hsp70 binds to Leu (Leu-640) immediately preceding the conserved turn motif autophosphorylation site (Thr-641) in PKC betaII. Co-immunoprecipitation experiments reveal that mutation of Leu-640 to Gly decreases the interaction of Hsp70 with PKC betaII. This weakened interaction between Hsp70 and the mutant PKCs results in accumulation of dephosphorylated PKC in the detergent-insoluble fraction of cells. In addition, the Hsp70-binding mutant is considerably more sensitive to down-regulation compared with WT PKC: disruption of Hsp70 binding leads to accelerated dephosphorylation and enhanced ubiquitination of mutant PKC upon phorbol ester treatment. Last, pulse-chase experiments demonstrate that Hsp70 preferentially binds the species of mature PKC that has become dephosphorylated compared with the newly synthesized protein that has yet to be phosphorylated. Thus, Hsp70 binds a hydrophobic residue preceding the turn motif, protecting PKC from down-regulation and sustaining the signaling lifetime of the kinase.

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    • "PKA, PKB or Akt, and PKC) share a conserved turn motif that enables enzymatic activity when phosphorylated (Newton, 2003). This motif is also a binding domain for the Hsc/ Hsp70 chaperones (Gao and Newton, 2006). Binding of these chaperones is crucially important, because ABC kinases expose their turn motif to phosphatases when they are catalytically active. "
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    ABSTRACT: Atypical PKC (ι/λ and ζ) is a key player in the acquisition of epithelial polarity and participates in other signaling cascades including NF-kB control. This kinase is post-translationally regulated by Hsp70-mediated refolding. Previous work has shown that such a chaperoning activity is specifically localized on keratin intermediate filaments. This work was performed with the goal of identifying the molecule(s) blocking Hsp70 activity on keratin filaments in inflammation. A transcriptional screen allowed us to focus on BAG-1, a multi-functional protein which assists Hsp70 in nucleotide exchange, but also blocks its activity at higher concentrations. We found the BAG-1M isoform upregulated 3 fold under TNFα stimulation in Caco-2 cells and up to 6 fold in mouse enterocytes under DSS colitis. BAG-1M but not other isoforms, was found to copurify with intermediate filaments and block Hsp70 activity in the keratin fraction but not in the soluble fraction within the range of concentrations found in epithelial cells. BAG-1M constitutive expression decreased p-aPKC. BAG-1 knockdown, conversely, blocked the TNFα-induced decreased levels of p-aPKC. We conclude that BAG-1M mediates Hsp70 inhibition downstream of NF-kB.
    Journal of Cell Science 05/2014; 127(16). DOI:10.1242/jcs.151084 · 5.43 Impact Factor
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    • "The finding that several HSP could not rescue these defects furthermore suggests that the unfolding is likely rather subtle. In line, SCA14-mutant PKCc proteins were found to be resistant to the ubiquitin– proteasome system degradation (Seki et al. 2007), and phosphorylated PKC proteins could not be properly downregulated (Gao and Newton 2006). These findings imply that proteasomes cannot handle phosphorylated PKCc molecules, as was shown for PKCc-V138E, because they are not properly chaperoned into a folding/degradation competent Fig. 7 The effect of phosphoinositide-dependent protein kinase 1 (PDK1) over-expression on the accumulation of spinocerebellar ataxia type 14 (SCA14)-mutant protein kinase C (PKC)c-V138E in Tritoninsoluble fraction. "
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    ABSTRACT: The protein kinase C γ (PKCγ) undergoes multi-step activation and participates in various cellular processes in Purkinje cells. Perturbations in its phosphorylation state, conformation or localization can disrupt kinase signaling, such as in Spinocerebellar ataxia type 14 (SCA14) that is caused by missense mutations in PRKCG encoding for PKCγ. We previously showed that SCA14 mutations enhance PKCγ membrane translocation upon stimulation due to an altered protein conformation. Since the faster translocation did not result in an increased function, we examined how SCA14 mutations induce this altered conformation of PKCγ and what the consequences of this conformational change are on PKCγ life cycle. Here, we show that SCA14-related PKCγ-V138E exhibits an exposed C-terminus as shown by FRET-FLIM microscopy in living cells, indicative of its partial unfolding. This conformational change was associated with faster PMA-induced translocation and accumulation of fully phosphorylated PKCγ in the insoluble fraction, which could be rescued by co-expressing PDK1 kinase, that normally triggers PKCγ autophosphorylation. We propose that the SCA14 mutation V138E causes unfolding of the C1B domain and exposure of the C-terminus of the PKCγ-V138E molecule, resulting in a decrease of functional kinase in the soluble fraction. This article is protected by copyright. All rights reserved.
    Journal of Neurochemistry 10/2013; 128(5). DOI:10.1111/jnc.12491 · 4.28 Impact Factor
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    • "Once PKC is dephosphorylated, it becomes Triton X-100 insoluble and binds to Hsc/Hsp70 chaperones. Then PKC either can be ubiquitinylated and degraded or may be " rescued " through Hsp70–mediated refolding and subsequent rephosphorylation (Gao and Newton, 2006). We recently showed that the same principle of enhanced dephosphorylation by activity applies to PKCι, which became the basis for the biochemical rescue assay (Mashukova et al., 2009). "
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    ABSTRACT: Phosphorylation of the activation domain of protein kinase C (PKC) isoforms is essential to start a conformational change that results in an active catalytic domain. This activation is necessary not only for newly synthesized molecules, but also for kinase molecules that become dephosphorylated and need to be refolded and rephosphorylated. This "rescue" mechanism is responsible for the maintenance of the steady-state levels of atypical PKC (aPKC [PKCι/λ and ζ]) and is blocked in inflammation. Although there is consensus that phosphoinositide-dependent protein kinase 1 (PDK1) is the activating kinase for newly synthesized molecules, it is unclear what kinase performs that function during the rescue and where the rescue takes place. To identify the activating kinase during the rescue mechanism, we inhibited protein synthesis and analyzed the stability of the remaining aPKC pool. PDK1 knockdown and two different PDK1 inhibitors-BX-912 and a specific pseudosubstrate peptide-destabilized PKCι. PDK1 coimmunoprecipitated with PKCι in cells without protein synthesis, confirming that the interaction is direct. In addition, we showed that PDK1 aids the rescue of aPKC in in vitro rephosphorylation assays using immunodepletion and rescue with recombinant protein. Surprisingly, we found that in Caco-2 epithelial cells and intestinal crypt enterocytes PDK1 distributes to an apical membrane compartment comprising plasma membrane and apical endosomes, which, in turn, are in close contact with intermediate filaments. PDK1 comigrated with the Rab11 compartment and, to some extent, with the transferrin compartment in sucrose gradients. PDK1, pT555-aPKC, and pAkt were dependent on dynamin activity. These results highlight a novel signaling function of apical endosomes in polarized cells.
    Molecular biology of the cell 03/2012; 23(9):1664-74. DOI:10.1091/mbc.E11-12-0988 · 4.47 Impact Factor
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