Abstract
The life cycle of protein kinase C (PKC) is tightly controlled by mechanisms that mature the enzyme, sustain the activation-competent enzyme, and degrade the enzyme. Here we show that a conserved PXXP motif (Kannan, N., Haste, N., Taylor, S. S., and Neuwald, A. F. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 1272-1277), in the C-terminal tail of AGC (c-AMP-dependent protein kinase/protein kinase G/protein kinase C) kinases, controls the processing phosphorylation of conventional and novel PKC isozymes, a required step in the maturation of the enzyme into a signaling-competent species. Mutation of both Pro-616 and Pro-619 to Ala in the conventional PKC betaII abolishes the phosphorylation and activity of the kinase. Co-immunoprecipitation studies reveal that conventional and novel, but not atypical, PKC isozymes bind the chaperones Hsp90 and Cdc37 through a PXXP-dependent mechanism. Inhibitors of Hsp90 and Cdc37 significantly reduce the rate of processing phosphorylation of PKC. Of the two C-terminal sites processed by phosphorylation, the hydrophobic motif, but not the turn motif, is regulated by Hsp90. Overlay of purified Hsp90 onto a peptide array containing peptides covering the catalytic domain of PKC betaII identified regions surrounding the PXXP segment, but not the PXXP motif itself, as major binding determinants for Hsp90. These Hsp90-binding regions, however, are tethered to the C-terminal tail via a "molecular clamp" formed between the PXXP motif and a conserved Tyr (Tyr-446) in the alphaE-helix. Disruption of the clamp by mutation of the Tyr to Ala recapitulates the phosphorylation defect of mutating the PXXP motif. These data are consistent with a model in which a molecular clamp created by the PXXP motif in the C-terminal tail and determinants in the alphaE-helix of the catalytic domain allows the chaperones Hsp90 and Cdc37 to bind newly synthesized PKC, a required event in the processing of PKC by phosphorylation.
Highlights
C-terminal Tail of protein kinase C (PKC) II Is Necessary for Catalytic Activity—A conserved PXXP motif in the C-terminal tail of AGC kinases has been proposed to be an important site for allosteric regulation of the catalytic domain (Fig. 1, A and B) [36]
The asterisk denotes the position of fully phosphorylated PKC, and the dash denotes the position of unphosphorylated PKC
Binding of these chaperones is dependent upon a molecular clamp that is formed between a conserved PXXP motif in the C-terminal tail of PKC and determinants in the ␣E helix of the catalytic domain
Summary
Heat shock protein; PKC, protein kinase C; PKA, cAMP-dependent protein kinase; AGC, c-AMP-dependent protein kinase/protein kinase G/protein kinase C; PDK-1, phosphoinositide-dependent kinase 1; Cdc, cell division cycle 37; mTOR, mammalian target of rapamycin; RFP, red fluorescent protein; YFP, yellow fluorescent protein; PDBu, phorbol-12,13dibutyrate; PMA, phorbol 12-myrstate 13-acetate; 17-AAG, 17-(allylamino)-17demethoxygeldanamycin; MG-132, cabobenzoxyl-L-leucyl-L-leucyl-leucinal; DMEM, Dulbecco’s modified Eagle’s medium; WT, wild type. In addition to containing the key regulatory phosphorylation sites and docking the upstream kinase PDK-1, the C-terminal tail contains key conserved motifs, found within all AGC kinases, that facilitate ATP binding, promote substrate binding, and structure the catalytic core [36]. One such motif comprises the segment PXXP; this motif makes key contacts with the catalytic core, where it is important for modulating movement of the catalytic domain [36]. Our data support a model in which the PXXP motif participates in an intramolecular clamp with determinants in the ␣E helix of the kinase core, by providing a recognition surface for Hsp to bind and facilitate the maturation of PKC, a required step in the processing of the enzyme
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