title: The turn motif is a phosphorylation switch that regulates the binding of Hsp70 to protein kinase C.
abstract: Heat shock proteins play central roles in ensuring the correct folding and maturation of cellular proteins. Here we show that the heat shock protein Hsp70 has a novel role in prolonging the lifetime of activated protein kinase C. We identified Hsp70 in a screen for binding partners for the carboxyl terminus of protein kinase C. Co-immunoprecipitation experiments revealed that Hsp70 specifically binds the unphosphorylated turn motif (Thr(641) in protein kinase C beta II), one of three priming sites phosphorylated during the maturation of protein kinase C family members. The interaction of Hsp70 with protein kinase C can be abolished in vivo by co-expression of fusion proteins encoding the carboxyl terminus of protein kinase C or the carboxyl terminus of Hsp70. Pulse-chase experiments reveal that Hsp70 does not regulate the maturation of protein kinase C: the rate of processing by phosphorylation is the same in the presence or absence of disrupting constructs. Rather, Hsp70 prolongs the lifetime of mature protein kinase C; disruption of the interaction promotes the accumulation of matured and then dephosphorylated protein kinase C in the detergent-insoluble fraction of cells. Furthermore, studies with K562 cells reveal that disruption of the interaction with Hsp70 slows the protein kinase C beta II-mediated recovery of cells from PMA-induced growth arrest. Last, we show that other members of the AGC superfamily (Akt/protein kinase B and protein kinase A) also bind Hsp70 via their unphosphorylated turn motifs. Our data are consistent with a model in which Hsp70 binds the dephosphorylated carboxyl terminus of mature protein kinase C, thus stabilizing the protein and allowing re-phosphorylation of the enzyme. Disruption of this interaction prevents re-phosphorylation and targets the enzyme for down-regulation.
authors.affiliation: Department of Pharmacology, University of California, La Jolla, California 92093-0640, USA.
authors.forename: Tianyan
authors.initials: T
authors.lastname: Gao
authors.forename: Alexandra C
authors.initials: AC
authors.lastname: Newton
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020014:K562 Cells; D007700:Kinetics; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017354:Point Mutation; D011493:Protein Kinase C / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D064546:Protein Kinase C beta; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013912:Threonine; D014162:Transfection
chemical_list: D018840:HSP70 Heat-Shock Proteins; D007527:Isoenzymes; D011994:Recombinant Proteins; D013912:Threonine; D005982:Glutathione Transferase; D011493:Protein Kinase C; D064546:Protein Kinase C beta