Functional transformation of the chromatoid body in mouse spermatids requires testis-specific serine/threonine kinases

Shang, Peng; Baarends, Willy M.; Hoogerbrugge, Jos W.; Ooms, M. P.; Cappellen, Wiggert A. van; Jong, A.A.W. de; Dohle, Gert R.; Eenennaam, Hans van; Gossen, Jan A.; Grootegoed, J. Anton

doi:10.1242/jcs.059949

Cited by 87 publications

(126 citation statements)

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“…Amongst the identified proteins was the testis-specific Ser/Thr kinase TSSK1, which is essential for spermatogenesis in the mouse (Xu et al 2008, Shang et al 2010. Herein, we describe experiments that demonstrate a clear biochemical link between TSSK1, TSKS and PPP1CC2 in the mouse testis.…”

Section: Introductionmentioning

confidence: 88%

“…Guinea pig anti-TSKS and TSSK1 antibodies (Shang et al 2010) were used at dilutions of 1:500 for western blotting in cell lysate samples and of 1:5000 using purified fusion proteins with goat anti-guinea pig HRP secondary antibody (Jackson Immunoreagents, West Grove, PA, USA) at a dilution of 1:5000. For immunohistochemistry, the same anti-TSKS and TSSK1 primary antibodies were used at a dilution of 1:500, with Cy3-conjugated AffiniPure goat anti-guinea pig IgG (Jackson Immunoreagents) at a dilution of 1:5000.…”

Section: Antibodiesmentioning

confidence: 99%

“…For immunohistochemistry, the same anti-TSKS and TSSK1 primary antibodies were used at a dilution of 1:500, with Cy3-conjugated AffiniPure goat anti-guinea pig IgG (Jackson Immunoreagents) at a dilution of 1:5000. Guinea pig anti-TSSK2 (Shang et al 2010) was used at a dilution of 1:2000 for western blotting under the secondary antibody conditions used for other guinea pig antibodies listed above. Rabbit anti-TSKS (Shang et al 2010) was used for western blotting at a dilution of 1:500 with anti-rabbit IgG HRP-linked antibody (Cell Signalling Technology, Beverly, MA, USA) at a dilution of 1:5000.…”

Section: Antibodiesmentioning

confidence: 99%

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PPP1CC2 can form a kinase/phosphatase complex with the testis-specific proteins TSSK1 and TSKS in the mouse testis

MacLeod¹,

Shang²,

Booth³

et al. 2014

Reproduction

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The mouse protein phosphatase gene Ppp1cc is essential for male fertility, with mutants displaying a failure in spermatogenesis including a widespread loss of post-meiotic germ cells and abnormalities in the mitochondrial sheath. This phenotype is hypothesized to be responsible for the loss of the testis-specific isoform PPP1CC2. To identify PPP1CC2-interacting proteins with a function in spermatogenesis, we carried out GST pull-down assays in mouse testis lysates. Amongst the identified candidate interactors was the testis-specific protein kinase TSSK1, which is also essential for male fertility. Subsequent interaction experiments confirmed the capability of PPP1CC2 to form a complex with TSSK1 mediated by the direct interaction of each with the kinase substrate protein TSKS. Interaction between PPP1CC2 and TSKS is mediated through an RVxF docking motif on the TSKS surface. Phosphoproteomic analysis of the mouse testis identified a novel serine phosphorylation site within the TSKS RVxF motif that appears to negatively regulate binding to PPP1CC2. Immunohistochemical analysis of TSSK1 and TSKS in the Ppp1cc mutant testis showed reduced accumulation to distinct cytoplasmic foci and other abnormalities in their distribution consistent with the loss of germ cells and seminiferous tubule disorganization observed in the Ppp1cc mutant phenotype. A comparison of Ppp1cc and Tssk1/2 knockout phenotypes via electron microscopy revealed similar abnormalities in the morphology of the mitochondrial sheath. These data demonstrate a novel kinase/phosphatase complex in the testis that could play a critical role in the completion of spermatogenesis.

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Section: Introductionmentioning

confidence: 88%

Section: Antibodiesmentioning

confidence: 99%

Section: Antibodiesmentioning

confidence: 99%

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PPP1CC2 can form a kinase/phosphatase complex with the testis-specific proteins TSSK1 and TSKS in the mouse testis

MacLeod¹,

Shang²,

Booth³

et al. 2014

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“…TSSK1 and -2 are the largest members in the family, with C-ter-minal kinase domain extensions of 94 and 87 amino acids, respectively. The targeted disruption of TSSK1/TSSK2 (double knock-out) in mice resulted in abnormal spermatid development, and the observed phenotype was associated with structural defects in mitochondrial sheath formation and the premature loss of the chromatoid body-derived ring structure in spermatids (10). Similarly, the development of spermatids was also impaired in TSSK6 knock-out animals and resulted in the production of morphologically abnormal sperm (11).…”

mentioning

confidence: 99%

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

Jha

Coleman

Wong

et al. 2013

Journal of Biological Chemistry

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Background: Testis-specific serine/threonine kinases (TSSKs) are expressed in spermatids and are essential for male fertility. Results: HSP90 inhibition results in increased ubiquitination and degradation of TSSKs and blocks catalytic activation of TSSK4 and -6. Conclusion: The TSSK family of kinases is stabilized and activated by HSP90. Significance: HSP90 may play a critical role in differentiation of spermatids and male fertility.

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“…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)(8)(9)(10)(11)(12)(13).…”

mentioning

confidence: 99%

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

Jha

Wong

Zerfas

et al. 2010

Journal of Biological Chemistry

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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...

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Functional transformation of the chromatoid body in mouse spermatids requires testis-specific serine/threonine kinases

Cited by 87 publications

References 61 publications

PPP1CC2 can form a kinase/phosphatase complex with the testis-specific proteins TSSK1 and TSKS in the mouse testis

PPP1CC2 can form a kinase/phosphatase complex with the testis-specific proteins TSSK1 and TSKS in the mouse testis

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

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

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