Invariant Leu preceding turn motif phosphorylation site controls the interaction of protein kinase C with Hsp70.
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.
Gao
Tianyan
T
Department of Pharmacology, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.
Newton
Alexandra C
AC
D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology / Q000378:metabolism; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D005998:Glycine / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D007930:Leucine / Q000737:chemistry*; D051379:Mice; D010766:Phosphorylation; D011493:Protein Kinase C / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D064546:Protein Kinase C beta; D017434:Protein Structure, Tertiary
D018840:HSP70 Heat-Shock Proteins; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D007930:Leucine; D005998:Glycine