Evidence that protein phosphatase 5 functions to negatively modulate the maturation of the Hsp90-dependent heme-regulated eIF2alpha kinase.
The maturation and activation of newly synthesized molecules of the heme-regulated inhibitor of protein synthesis (HRI) in reticulocytes require their functional interaction with Hsp90. In this report, we demonstrate that protein phosphatase 5 (PP5), a previously documented component of the Hsp90 chaperone machine, is physically associated with HRI maturation intermediates. The interaction of PP5 with HRI is mediated through Hsp90, as mutants of PP5 that do not bind Hsp90 do not interact with HRI. PP5 was also present in Hsp90 heterocomplexes with another Hsp90 cohort, p50(cdc37), and expression of newly synthesized HRI enhanced the amount of p50(cdc37) associated with Hsp90/PP5-HRI heterocomplexes. The functional significance of the interaction of PP5 with Hsp90-HRI heterocomplexes was examined by characterizing the effects of compounds that impact PP5 activity in vitro. The protein phosphatase inhibitors okadaic acid and nodularin enhanced the kinase activity of HRI when applied during HRI maturation/activation, while the PP5 activators arachidonic and linoleic acid repressed HRI activity when applied during HRI maturation/activation. However, application of these compounds after HRI's "transformation" to an Hsp90-independent form did not similarly impact HRI's kinase activity. Furthermore, the Hsp90 inhibitor geldanamycin negated the effects of phosphatase inhibitors on HRI maturation/activation. The finding that PP5 downregulates an Hsp90-dependent process supports models for regulated Hsp90 function and describes a novel potential substrate for PP5 function in vivo.
Shao
Jieya
J
Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK 74078-3035, USA.
Hartson
Steven D
SD
Matts
Robert L
RL
D000818:Animals; D016718:Arachidonic Acid / Q000494:pharmacology; D016227:Benzoquinones; D018797:Cell Cycle Proteins / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D029721:Drosophila Proteins; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D047029:Lactams, Macrocyclic; D046911:Macromolecular Substances; D018832:Molecular Chaperones / Q000378:metabolism*; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019319:Okadaic Acid / Q000494:pharmacology; D010456:Peptides, Cyclic / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D011809:Quinones / Q000494:pharmacology; D011817:Rabbits; D012156:Reticulocytes / Q000187:drug effects / Q000378:metabolism; D019892:eIF-2 Kinase / Q000378:metabolism*
D016227:Benzoquinones; D018797:Cell Cycle Proteins; D029721:Drosophila Proteins; D004791:Enzyme Inhibitors; D005231:Fatty Acids, Unsaturated; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D046911:Macromolecular Substances; D018832:Molecular Chaperones; D009687:Nuclear Proteins; D010456:Peptides, Cyclic; D011809:Quinones; C063998:nodularin; D019319:Okadaic Acid; D016718:Arachidonic Acid; D019892:eIF-2 Kinase; D010749:Phosphoprotein Phosphatases; C089641:protein phosphatase 5; C001277:geldanamycin