Identification of a novel HSP70-binding cochaperone critical to HSP90-mediated activation of small serine/threonine kinase.
We previously reported the identification of small serine/threonine kinase (SSTK) that is expressed in postmeiotic germ cells, associates with HSP90, and is indispensable for male fertility. Sperm from SSTK-null mice cannot fertilize eggs in vitro and are incapable of fusing with eggs that lack zona pellucida. Here, using the yeast two-hybrid screen, we have discovered a novel SSTK-interacting protein (SIP) that is expressed exclusively in testis. The gene encoding SIP is restricted to mammals and encodes a 125-amino acid polypeptide with a predicted tetratricopeptide repeat domain. SIP is co-localized with SSTK in the cytoplasm of spermatids as they undergo restructuring and chromatin condensation, but unlike SSTK, is not retained in the mature sperm. SIP binds to SSTK with high affinity (K(d) ∼10 nM), and the proteins associate with each other when co-expressed in cells. In vitro, SIP inhibited SSTK kinase activity, whereas the presence of SIP in cells resulted in enzymatic activation of SSTK without affecting Akt or MAPK activity. SIP was found to be associated with cellular HSP70, and analyses with purified proteins revealed that SIP directly bound HSP70. Importantly, SSTK recruited SIP onto HSP90, and treatment of cells with the specific HSP90 inhibitor, 17-allylamino-17-demethoxygeldanamycin, completely abolished SSTK catalytic activity. Hence, these findings demonstrate that HSP90 is essential for functional maturation of the kinase and identify SIP as a cochaperone that is critical to the HSP90-mediated activation of SSTK.
Jha
Kula N
KN
Laboratory of Chemistry, Division of Therapeutic Proteins, Center for Drug Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892, USA.
Wong
Lily
L
Zerfas
Patricia M
PM
De Silva
Rukman S
RS
Fan
Ying-Xin
YX
Spiridonov
Nikolay A
NA
Johnson
Gibbes R
GR
D000818:Animals; D001483:Base Sequence; D016227:Benzoquinones / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D008297:Male; D051379:Mice; D008817:Mice, Mutant Strains; D008969:Molecular Sequence Data; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D013094:Spermatozoa / Q000378:metabolism*; D013737:Testis / Q000378:metabolism*
D016227:Benzoquinones; C111922:Cacybp protein, mouse; D002135:Calcium-Binding Proteins; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; C112765:tanespimycin; D017346:Protein Serine-Threonine Kinases; C501083:Tssk6 protein, mouse