Abstract
We have cloned the entire coding region of a mouse germ cell-specific cDNA encoding a unique protein kinase whose catalytic domain contains only three consensus subdomains (I–III) instead of the normal 12. The protein possesses intrinsic Ser/Thr kinase activity and is exclusively expressed in haploid germ cells, localizing only in their nuclei, and was thus named Haspin (forhaploid germ cell-specific nuclearprotein kinase). Western blot analysis showed that specific antibodies recognized a protein ofM r 83,000 in the testis. Ectopically expressed Haspin was detected exclusively in the nuclei of cultured somatic cells. Even in the absence of kinase activity, however, Haspin caused cell cycle arrest at G1, resulting in growth arrest of the transfected somatic cells. In a DNA binding experiment, approximately one-half of wild-type Haspin was able to bind to a DNA-cellulose column, whereas the other half was not. In contrast, all of the deletion mutant Haspin that lacked autophosphorylation bound to the DNA column. Thus, the DNA-binding activity of Haspin may, in some way, be associated with its kinase activity. These observations suggest that Haspin has some critical roles in cell cycle cessation and differentiation of haploid germ cells. We have cloned the entire coding region of a mouse germ cell-specific cDNA encoding a unique protein kinase whose catalytic domain contains only three consensus subdomains (I–III) instead of the normal 12. The protein possesses intrinsic Ser/Thr kinase activity and is exclusively expressed in haploid germ cells, localizing only in their nuclei, and was thus named Haspin (forhaploid germ cell-specific nuclearprotein kinase). Western blot analysis showed that specific antibodies recognized a protein ofM r 83,000 in the testis. Ectopically expressed Haspin was detected exclusively in the nuclei of cultured somatic cells. Even in the absence of kinase activity, however, Haspin caused cell cycle arrest at G1, resulting in growth arrest of the transfected somatic cells. In a DNA binding experiment, approximately one-half of wild-type Haspin was able to bind to a DNA-cellulose column, whereas the other half was not. In contrast, all of the deletion mutant Haspin that lacked autophosphorylation bound to the DNA column. Thus, the DNA-binding activity of Haspin may, in some way, be associated with its kinase activity. These observations suggest that Haspin has some critical roles in cell cycle cessation and differentiation of haploid germ cells. Maturation of male germ cells in mammals involves numerous structural and functional changes that are precisely timed. These complex processes, known collectively as spermatogenesis, may be represented by the following three major events: proliferation and differentiation of spermatogonia, meiotic events at prophase of spermatocytes, and drastic morphological change during differentiation from the haploid round spermatids to sperm (1Russell L.D. Ettlin R.A. Sinha H.A.P. Clegg E.D. Russell L.D. Ettlin R.A. Sinha H.A.P. Clegg E.D. Histological and Histopathological Evaluation of the Testis. Cache River Press, FL1990: 1-38Google Scholar). To uncover the mechanism of spermatogenesis, many germ cell-specific molecules have been studied using various strategies (2Ikawa M. Wada I. Kominami K. Watanabe D. Toshimori K. Nishimune Y. Okabe M. Nature. 1997; 387: 607-611Crossref PubMed Scopus (247) Google Scholar, 3Kerr S.M. Vambrie S. McKay S.J. Cooke H.J. Mamm. Genome. 1994; 5: 557-565Crossref PubMed Scopus (24) Google Scholar, 4Tanaka H. Yoshimura Y. Nishina Y. Nozaki M. Nojima H. Nishimune Y. FEBS Lett. 1994; 355: 4-10Crossref PubMed Scopus (95) Google Scholar, 5Tsuchida J. Nishina Y. Wakabayashi N. Nozaki M. Sakai Y. Nishimune Y. Dev. Biol. 1998; 197: 67-76Crossref PubMed Scopus (26) Google Scholar). During haploid germ cell differentiation, or spermiogenesis, the round spermatid undergoes marked morphological change to become a sperm without cell division; and the nucleus is shaped, mitochondria are rearranged, the flagellum is developed, and the acrosome is generated (6Bellve A.R. O'Brien D.A. Hartmann J.F. Mechanism and Control of Animal Fertilization. Academic Press, New York1983: 55-237Google Scholar). Over this long period of time, ∼2 weeks for the mouse, no division of haploid germ cells occurs. Some of the regulatory proteins localized in the nucleus must participate in this precise regulation. It appears that there are at least two types of mechanisms at work in haploid germ cell-specific gene regulation: one involves the CRE, 1The abbreviations used are: CRE, cAMP response element; RACE, rapid amplification of cDNA ends; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; EGFP, enhanced green fluorescent protein; FACS, fluorescence-activated cell sorter and the other, as yet uncharacterized, does not. Several proteins are specifically expressed in the nuclei of haploid germ cells: transition protein (7Heidran M.A. Kozak C.A. Kistler W.S. Gene (Amst.). 1989; 75: 39-46Crossref PubMed Scopus (64) Google Scholar), protamine (8Johnson P. Peschon J.J. Yelick P.C. Palmiter R.D. Hecht N.B. Biochim. Biophys. Acta. 1988; 950: 45-53Crossref PubMed Scopus (105) Google Scholar), histone H1t (9Grimes Jr., S.R. van Wert J. Wolfe S.A. Mol. Biol. Rep. 1997; 24: 175-184Crossref PubMed Scopus (14) Google Scholar), zinc finger proteins (10Cunliffe V. Koopman P. McLaren A. Trowsdale J.A. EMBO J. 1990; 9: 197-205Crossref PubMed Scopus (75) Google Scholar), testis-specific HMG (11Boissonneault G. Lau Y.F. Mol. Cell. Biol. 1993; 13: 4323-4330Crossref PubMed Scopus (28) Google Scholar), lamin B3 (12Furukawa K. Hotta Y. EMBO J. 1993; 12: 97-106Crossref PubMed Scopus (205) Google Scholar), and CREMτ (13Foulkes N.S. Mellstrom B. Benusiglio E. Sassone-Corsi P. Nature. 1992; 355: 80-84Crossref PubMed Scopus (409) Google Scholar). Through knockout of theCremτ gene in mice, CREMτ capable of binding to the sequence of CRE has been shown to play important roles in the regulation of spermiogenesis (14Peschon J.J. Behringer R.R. Brinster R.L. Palmiter R.D. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5316-5319Crossref PubMed Scopus (176) Google Scholar, 15Blendy J.A. Kaestner K.H. Weinbauer G.F. Nieschlag E. Schutz G. Nature. 1996; 380: 162-165Crossref PubMed Scopus (466) Google Scholar). In contrast, some of the proteins specifically expressed in haploid germ cells do not have any CRE motifs in the promoter region (16Wisniewski J. Malezewski M. Krawczyk Z. Gedamu L. Eur. J. Biochem. 1993; 212: 137-143Crossref PubMed Scopus (23) Google Scholar), implying that some other regulatory mechanism exists. We have isolated many cDNA clones specifically expressed in germ cells using a subtracted cDNA library prepared from supporting cells of mutant testes and the wild-type testis (4Tanaka H. Yoshimura Y. Nishina Y. Nozaki M. Nojima H. Nishimune Y. FEBS Lett. 1994; 355: 4-10Crossref PubMed Scopus (95) Google Scholar). Using a partial cDNA clone previously obtained, we have cloned the entire coding region and characterized a novel gene encoding a protein with various well known motifs. The protein, which we named Haspin (haploid germ cell-specific nuclearprotein kinase), is specifically expressed in haploid germ cells, localizes in nuclei of round spermatids, binds to DNA, and has Ser/Thr protein kinase activity. Since transfection ofhaspin cDNA into cultured somatic cells caused cessation of cell proliferation, Haspin could be involved in regulation of proliferative activity as well as specific gene expression in haploid germ cells. Total RNAs were extracted by the guanidine thiocyanate/CsTFA method (17Okayama H. Kawaich M. Brownstein M. Lee F. Yokota T. Araki K. Methods Enzymol. 1987; 154: 3-28Crossref PubMed Scopus (286) Google Scholar) from the testes of adult wild-type C57BL/6 mice and 4-month-old W/Wv mutant mice lacking germ cells (4Tanaka H. Yoshimura Y. Nishina Y. Nozaki M. Nojima H. Nishimune Y. FEBS Lett. 1994; 355: 4-10Crossref PubMed Scopus (95) Google Scholar). The corresponding cDNA libraries were prepared as described by Gubler and Hoffmann (18Gubler U. Hoffmann B.J. Gene (Amst.). 1983; 25: 263-269Crossref PubMed Scopus (3076) Google Scholar), with some modifications. Prepared cDNA fragments were directionally inserted between NotI (dephosphorylated) andBglII sites of the pAP3neo vector (4Tanaka H. Yoshimura Y. Nishina Y. Nozaki M. Nojima H. Nishimune Y. FEBS Lett. 1994; 355: 4-10Crossref PubMed Scopus (95) Google Scholar). The ligated DNAs were electroporated into MC1061A cells as described previously (19Kobori M. Nojima H. Nucleic Acids Res. 1993; 21: 2782Crossref PubMed Scopus (29) Google Scholar). The complexities of the cDNA libraries obtained were ∼6 × 106 colony-forming units in both cases. A germ cell-specific cDNA library was generated by subtracting cDNAs of mutant (W/WV) testis that contains no germ cells from wild-type testis cDNAs (4Tanaka H. Yoshimura Y. Nishina Y. Nozaki M. Nojima H. Nishimune Y. FEBS Lett. 1994; 355: 4-10Crossref PubMed Scopus (95) Google Scholar). Plasmid DNA of each clone randomly picked from the subtracted cDNA library was subjected to cDNA dot-blot analysis. As a probe to select testicular germ cell-specific cDNA clones, RNAs of testis cDNA libraries of wild-type and mutant mice were generated with T7 RNA polymerase and labeled with the hapten digoxigenin. To clone the complete cDNA ofgsg2, a testis-specific partial cDNA obtained previously by screening the subtracted library (4Tanaka H. Yoshimura Y. Nishina Y. Nozaki M. Nojima H. Nishimune Y. FEBS Lett. 1994; 355: 4-10Crossref PubMed Scopus (95) Google Scholar), a library of Escherichia coli MC1061A cells carrying the adult wild-type testis cDNAs was diluted to seed at 1 × 105 colony-forming units on a nitrocellulose filter placed on an LB plate. After incubation at 37 °C, grown colonies were transferred to two nylon replica filters and