Identification of a Novel HSP70-binding Cochaperone Critical to HSP90-mediated Activation of Small Serine/Threonine Kinase

Abstract

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 ϳ10 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.Protein phosphorylation plays important roles during spermatogenesis (1, 2). A number of protein kinases are expressed in testis, and several of them are essential for male fertility (3). For example, disruption of the Camk4 which is involved in postmeiotic chromatin condensation, results in impaired spermiogenesis and male sterility (4). Targeted deletion of the casein kinase 2␣Ј catalytic subunit leads to oligozoospermia and extensive degeneration of germ cells at all stages of spermatogenesis (5), and Cdk2 is essential for completing mitotic division in germ cells (6). Three members of the testis-specific serine threonine kinase (TSSK) 2 family, namely TSSK1, TSSK2, and the small serine/threonine kinase (SSTK, also known as TSSK6), are expressed postmeiotically and are essential for male fertility (7-13).SSTK is one of the smallest protein kinases, consists only of N-and C-lobes of a kinase catalytic domain, and forms stable associations with heat shock protein (HSP) 70 and 90 (7). Targeted deletion of Sstk in mice resulted in male infertility without any other visible somatic defects. SSTK-null sperm could not fertilize eggs in vitro and are incapable of fusing with eggs that lack zona pellucida (9). SSTK expression first appears in elongating spermatids as they undergo cellular remodeling and chromatin condensation, and the kinase is retained in mature sperm. However, the in vivo substrate(s) for SSTK, the mechanism of activation, and the specific function that SSTK performs in spe...

Heat Shock Protein 90 Functions to Stabilize and Activate the Testis-specific Serine/Threonine Kinases, a Family of Kinases Essential for Male Fertility

“…Co-chaperones play an integral role in the specificity of HSP90 for client proteins. Recently, we reported the identification and characterization of the HSP70 binding co-chaperone SIP/TSACC that is expressed exclusively in spermatids (33). SIP binds to TSSK6 and facilitates the HSP90-mediated activation of TSSK6.…”

“…Protein Expression in 293T and COS-7 Cells-Myc epitopetagged TSSKs and CDK2 cDNA construct was transfected in 293T cells as described earlier (33). Transfections of Myc epitope-tagged TSSKs, CDK2, and HA-tagged ubiquitin in COS-7 cells were carried out using the DEAE-dextran chloroquine technique (36).…”

“…Protein Kinase Assay-Kinase reactions were performed as described previously (33). Briefly, the reaction was carried out at room temperature for 30 min in buffer that contained 25 mM HEPES, pH 7.4, 10 mM MgCl 2 , 10 mM MnCl 2 , 2 mM EGTA, 30 M ATP, 10 Ci of [␥-32 P]ATP, and a protein substrate.…”

“…Western Blotting and Immunoprecipitation-Western blotting and immunoprecipitation (IP) were performed as described previously (33). Myc IP from denatured lysate for the ubiquitination assay, as depicted in Fig.…”

“…Recently we identified an HSP70 binding co-chaperone named small serine/threonine kinase (SSTK)-interacting protein (SIP; TSSK6-activating co-chaperone (TSACC)) that facilitates the HSP90-mediated activation of TSSK6 (33). However, it remained to be determined whether the role of HSP90 in activation was limited to TSSK6 or if the other TSSKs were also regulated by HSP90.…”