Identification and Characterization of a Haploid Germ Cell-specific Nuclear ProteinKinase (Haspin) in Spermatid Nuclei and Its Effects on Somatic Cells (1999)

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 at were to two and by in the following at in and in and and in After at for the were and the was A probe was prepared with a using a of the cDNA (4Tanaka H. Yoshimura Y. Nishina Y. Nozaki M. Nojima H. Nishimune Y. FEBS Lett. 1994; 355: 4-10Crossref PubMed Scopus (95) Google Scholar). The were with the partial clone probe at for and sperm Several clones were isolated by screening × 106 To the of the we J. J. Nucleic Acids Res. PubMed Scopus Google Scholar, M.A. Proc. Natl. Acad. Sci. U. S. A. 1988; PubMed Scopus Google Scholar) using a of the cDNA was using of a from the sequence of which to of the cDNA and of mouse testis After of RNA with DNAs were by and ligated to with amplification of the region was using a corresponding to and a and an were with and and ligated to clones were and of adult mice were in and other somatic cells of the testes were prepared as described in M. H. K. J. K. M. S. K. A. Nishimune Y. Biol. 1998; PubMed Scopus Google Scholar). testes and of various in germ cell differentiation as and W/Wv were used J. Nishina Y. Wakabayashi N. Nozaki M. Sakai Y. Nishimune Y. Dev. Biol. 1998; 197: 67-76Crossref PubMed Scopus (26) Google Scholar). Total RNAs were extracted to the and by RNA were subjected to on a gel J. E. T. A Scholar). RNAs were to a nitrocellulose filter in was with cDNA prepared with the at for in a sperm DNA, and were in and at of the were detected with a F. S. A.R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) were with fluorescent and cycle from The were by a DNA and were for to the or The sequence of cDNA was by the of at and at from the sequence of Haspin were by and used for of a partial cDNA was into the vector Gene (Amst.). 1988; PubMed Scopus Google Scholar). proteins were coli by and with were obtained by of the by at in into New and with the by Western blot analysis. The was used for each prepared or cell of adult C57BL/6 mice or cultured cells were on with a and After the protein of each was by the protein of protein was subjected to as by Nature. PubMed Scopus Google Scholar), by The were with and for with and The were with in at for 1 with for three for each time, and with at for 1 After were by with in To testes were in and at of in were prepared by using a and were with at for was with with normal in and with diluted at a of with a were with diluted at and subjected to a by with in Some of the were with amplification of the cDNA coding region was using an for the of cDNA and a of the were with which to of the cDNA and with whose been in the and ligated at of the expression vector The clone was capable of the The expression vector with a deletion mutant of was by polymerase amplification using cDNA as a the following was the cDNA was into two in the region of the using the and a deletion was by polymerase and the two DNA fragments were to a complete deletion The sequence corresponding to and the of the as described were used for the DNA of the deletion mutant The sequence corresponding to and the of the as described were used for the DNA of the deletion mutant The two DNA fragments were into sites of to obtained the deletion mutant cDNA The cDNA obtained, the deletion mutant was ligated to the of the expression vector Y. Cell. PubMed Scopus Google Scholar) and Biol. PubMed Google Scholar) cells were transfected with expression and using H. Mol. Cell. Biol. 1987; PubMed Scopus Google Scholar) and cells were with and and in in the of cells, transfected cells were with the and After protein kinase the cells were with and with a a The expression vector without cDNA was used as a testes and transfected cells were with a of and PMSF, and the were at for at The was with protein at for 1 to binding normal or specific was at a and at for The were with protein at for 1 and The protein were three with the and two with kinase and and at for in of kinase with of The with were subjected to and to proteins were with a analysis was as described J.A. T. Methods Enzymol. 1983; PubMed Scopus Google Scholar) in the protein kinase of the at After transfection with expression vector on and cells were in and with After with cells were with The cell were with a fluorescence-activated cell sorter The between the Haspin protein and DNA was by an of the Haspin protein by DNA transfected with or cells were in and 1 were at for at and a were to with DNA-cellulose were with of and in 1 two with of of 1 The were and subjected to Western blot analysis with cDNA clones have been isolated by screening × 106 clones from a testis cDNA library with the probe that been previously cloned as a germ cell-specific cDNA (4Tanaka H. Yoshimura Y. Nishina Y. Nozaki M. Nojima H. Nishimune Y. FEBS Lett. 1994; 355: 4-10Crossref PubMed Scopus (95) Google Scholar). of the with of a long As no was of the we using prepared from the sequence and isolated Since a was of the sequence in all clones, we the to be the of the cDNA The complete sequence and its sequence are shown in The protein of from the at A for the sequence that no sequence has been The cDNA sequence was without a a region of two consensus at and The sequence of the cDNA showed the following well known motifs 1 a at R.A. Biochem. Sci. PubMed Scopus Google a at 1988; PubMed Scopus Google and of protein kinase consensus the region of Methods Enzymol. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar), which was in to kinase It many sites for protein sites for protein kinase A. K. H. Y. H. Y. Y. J. Biol. PubMed Google Scholar) at and a and protein kinase J. Biol. PubMed Google Scholar) at and sites for kinase Biochim. Biophys. Acta. 1990; PubMed Scopus Google Scholar) in Haspin It a region to K. T. K. K. T. Biochem. Biophys. Res. PubMed Scopus Google Scholar) at We named this protein blot analysis showed that was specifically detected in the testis as a major of expressed exclusively in germ cells the was not in somatic as the and was in the In both the and mutant mouse in which there are no germ cells J. Nishina Y. Wakabayashi N. Nozaki M. Sakai Y. Nishimune Y. Dev. Biol. 1998; 197: 67-76Crossref PubMed Scopus (26) Google Scholar), no was Western blot analysis with detected one with specifically in mouse testis with the of blot analysis. A with a was detected in the with this the protein was to be a no was detected in the by blot analysis. During male germ cell the was not detected in mouse testis of the to The change in the of the was to the for protamine that its expression in the testis is haploid In the of Western blot Haspin was detected in the and the with was with the expression Thus, that both and of the gene exclusively in haploid male germ cells and that the of gene expression is precisely during the of male germ expression of and protein in mice of RNA from mouse testes was used for blot analysis. the of and and of testicular protein from mice of was and to a Western blot analysis was with as described of Haspin in adult mouse testis showed that some germ cells somatic cells as and cells were The expression of the Haspin protein was detected in haploid round spermatids at 1 the as differentiation In spermatids at and in which was no was germ cells in the of the and and were These observations were in with the of Western blot that Haspin is a novel protein whose expression at the of haploid of Haspin was to the nuclei of round and of spermatids of the nucleus and acrosome of Haspin to change from the nucleus to a of the in the of the acrosome Haspin has a kinase catalytic domain that consensus kinase subdomains the consensus subdomains To the of we its kinase activity. testis were with of the was subjected to in kinase in the of and with The and the were both subjected to by Western blot analysis with using the proteins labeled with were detected by and Haspin proteins were detected by of r 83,000 and labeled with were detected These two to Haspin and the of In one was detected at r the was detected with all three be a specific associated with Haspin could be a of To that the kinase activity was intrinsic to a deletion of was into kinase is to bind The expression were to the wild-type and mutant Haspin each with green protein and and were transfected into cells. The expressed Haspin proteins were with Western blot analysis with one major r was detected for both the wild-type and deletion mutant Haspin proteins The of the transfected cell were subjected to in kinase in the of by Western blot analysis. The showed no on the deletion mutant lacking kinase I. analysis that both and were specifically that Haspin has intrinsic kinase activity To the of we have its and the of its expression on cultured cells. and cells were transfected with the cDNA of wild-type or deletion both proteins were localized exclusively in the nuclei of the cultured cells to the of Haspin in haploid spermatids In the cultured cells, however, in Haspin was to some in a the nucleus and transfected cells not we have the of Haspin on cell cycle cells were transfected with wild-type or deletion mutant After an period of cells were and into two cells and cells The cells were the cells could be as the for the cells capable of The cells in both mutant and wild-type showed the normal growth and In contrast, the cells showed cell cycle arrest at and Thus, the transfected cells capable of Haspin were not able to and all the cells from the The expression of deletion mutant Haspin lacking the kinase activity was to that of wild-type Haspin in transfected cells from the of Haspin not the deletion mutant a response the all of the cells mutant Haspin cell cycle arrest at whereas with wild-type Haspin showed cell cycle arrest It be that the protein the cell and and not the cell cycle or growth of the transfected cells not analysis of cells transfected with the and deletion mutant in expression vector The cells in the transfected with cDNA were into transfected and cells to of on and the cells were subjected to analysis. A was with to transfected cells from cells in the shown are changes in the DNA of transfected and cells transfection with and deletion mutant cDNAs in the Since both the and expression of Haspin were to nuclei, we the of Haspin with The binding of Haspin and mutant Haspin for DNA were by their by DNA from the cells transfected with the wild-type or mutant cDNA were to DNA-cellulose and proteins were with of After the proteins were detected by Western blot analysis with As shown in half the of the Haspin protein was from the and the was by the DNA column. all the bound Haspin was In contrast, all of the mutant Haspin in kinase activity was able to bind to the DNA and was with Thus, that the mutant Haspin lacking the autophosphorylation activity has for DNA the and suggest that Haspin is in at least two in of DNA-binding we have isolated various germ cell-specific cDNAs from a subtracted cDNA library of the mouse testis (4Tanaka H. Yoshimura Y. Nishina Y. Nozaki M. Nojima H. Nishimune Y. FEBS Lett. 1994; 355: 4-10Crossref PubMed Scopus (95) Google Scholar) and the roles of in The that Haspin is a haploid germ cell-specific protein expressed exclusively in spermatids from 1 to and as the spermatids Western blot analysis using three and capable of three in the Haspin showed one of r 83,000 exclusively in the testis. In a was detected in the with the protein must be a other Haspin no was detected with the other two not and no was in the by blot analysis. Haspin contains known motifs in the a of the protein kinase consensus sequence was protein to a of proteins a catalytic of motifs to both the and protein Methods Enzymol. PubMed Scopus Google J. PubMed Scopus Google Scholar). of the motifs involved in binding is in the of the Haspin protein Haspin has only three consensus motifs of the kinase motifs. To Haspin has kinase activity or we an in kinase The suggest that Haspin has kinase activity and its and the was by a deletion into without any sites and Haspin showed no or autophosphorylation protein kinase activity, thus that Haspin has intrinsic protein kinase activity. Haspin contains many sites for protein sites for protein kinase A. K. H. Y. H. Y. Y. J. Biol. PubMed Google Scholar), kinase Biochim. Biophys. Acta. 1990; PubMed Scopus Google Scholar), and and protein J. Biol. PubMed Google Scholar). It is that the activity of the of Haspin is from that of the We have shown that Haspin with DNA was between wild-type and mutant Haspin of which the was capable of autophosphorylation and the was not. The with DNA may some activity, and be involved in the a could be the region of using the consensus sequence 1988; PubMed Scopus Google Scholar), that Haspin with some other proteins to protein many proteins have been and to play roles in cell growth and differentiation T. M. K. H. S. Mol. Cell. Biol. 1998; PubMed Scopus Google Scholar). In haploid germ cells, a specific cAMP response protein is to some gene expression L. F. N.S. Sassone-Corsi P. V. K. in New New Scholar, Z. A.R. V. K. in New New Scholar). Haspin may with a to play a regulatory in gene Haspin has a R.A. Biochem. Sci. PubMed Scopus Google Scholar) at the sequence was expressed Haspin was exclusively in nuclei of or cells not Thus, the is to play an important in specific of Haspin in spermatid It is that the sequence in the region of Haspin for the specific in the the Haspin to in a implying its with some as expression of Haspin caused cell cycle arrest at in transfected and cells and cells not The of which was not to the of the S. M. U. Res. 1997; PubMed Scopus Google Scholar) in cultured cell was not to the kinase activity the or a was with the deletion mutant Haspin we the DNA-binding of Haspin using the DNA-cellulose there was a between the wild-type and mutant Haspin one-half of wild-type Haspin was bound to DNA, the was not and was from the In contrast, mutant Haspin bound to the DNA Thus, wild-type Haspin has for DNA by the of The region of Haspin as a DNA-binding and its binding be by In this way, autophosphorylation may participate in the regulation of cell cycle Since Haspin caused cell cycle arrest in somatic cells, may a in the testis. It be however, that Haspin expression was only in the haploid germ cells that were no able to In Haspin may participate in the of the cell cycle of Haspin has a region to at K. T. K. K. T. Biochem. Biophys. Res. PubMed Scopus Google Scholar) and has a region at its gene a which the expression of many K. B. Biol. 1996; PubMed Scopus Google Scholar). As Haspin does not have the and does not have the protein kinase Haspin in from Haspin has the domain at its and bind to Thus, is that Haspin is of testis-specific or a cell cycle regulatory of haploid germ cells. The gene has been to mouse M. H. S. H. J. Nozaki M. Yoshimura Y. Nojima H. Nishimune Y. Mamm. Genome. 1997; PubMed Scopus Google Scholar). A known mouse mutant to is the mutant which a to N. J. PubMed Scopus Google Scholar). corresponding gene in is the Haspin be a of germ cell are in to the of Haspin in testicular germ cell proliferation and We are to A. for