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

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 (for haploid germ cell-specific nuclear protein kinase). Western blot analysis showed that specific antibodies recognized a protein of M 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 G 1 , resulting in growth arrest of the transfected somatic cells. In a DNA binding experiment, approximately onehalf of wild-type Haspin was able to bind to a DNAcellulose 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.

In vitro production of fertile sperm from murine spermatogonial stem cell lines

“…This particular application of our method would break the boundary between germ and somatic cells, and open a new phase in germ cell research. 18 , Gsg2/Haspin-GFP 23 , and Acr-GFP [21][22] , were used to produce GS cells. The pCXN-GFP mice were provided by Dr. Okabe, Gsg2/Haspin-GFP mice were provided by Dr. Tanaka, and Acr-GFP mice were provided by RIKEN BRC through the National Bio-Resource Project of MEXT, Japan.…”

“…To confirm that GS cells are able to proceed up to or beyond the meiotic phase of spermatogenesis, we exploited 2 lines of transgenic mice, Acr-GFP 21,22 and Gsg2-GFP 23 , to establish GS cells from them. We confirmed that the GS cells derived from Acr-GFP (Acr-GS cells) and Gsg2-GFP (Gsg2-GS cells) expressed GFP at the expected phases of differentiation, mid-and end-meiotic, respectively, when transplanted in the testis in vivo (data not shown).…”

The Mitotic Protein Kinase Haspin and Its Inhibitors

“…The coded protein localizes in the nucleus of germ cells and showed kinase activity. It was subsequently renamed haploid germ cell-specific nuclear protein kinase (Haspin) [4]. Haspin mRNA is found in diploid cells of many proliferative tissues such as testis, thymus, bone marrow, and spleen as well as in many proliferative cell lines [5][6][7].…”

“…So far, the precise cellular localization of the endogenous protein could not be determined due to lack of immunofluorescence-specific antibodies to Haspin. However, several overexpression studies have reported localization of GFP-or Myc-tagged Haspin in different eukaryotic cell lines (HeLa, U-2 OS, Hek293, COS-7) using time-lapse video microscopy or immunofluorescence staining techniques or time-lapse video microscopy [4,13]. All these studies showed that Haspin localizes in discrete foci and nucleoli in the nucleus during interphase.…”

“…The fact that Haspin is an atypical ePK with a divergent structure may lead to the development of inhibitors with fewer side effects [4,5]. Haspin inhibitor CHR6494 described by Huertas et al showed antitumor activity in a xenograph mouse model [53].…”