Male Germ Cell-associated Kinase Research Articles

Androgen Receptor–Dependent PSA Expression in Androgen-Independent Prostate Cancer Cells Does Not Involve Androgen Receptor Occupancy of the PSA Locus

It is widely suspected that androgen-independent prostate cancer growth depends on androgen receptor signaling via ill-defined mechanisms. Prostate-specific antigen (PSA) expression is often used to measure androgen receptor activity in cells and prostate cancer progression in patients. In the present study, we have compared androgen receptor activity using PSA and human male germ cell-associated kinase (hMAK), as read-outs in androgen-dependent LNCaP and androgen-independent C4-2B cells. As expected, very little PSA and hMAK expression were detected in LNCaP cells in the absence of androgens, whereas substantial expression of PSA was observed only in C4-2B cells under the same conditions. The addition of dihydrotestosterone to the culture medium increased the expression of both genes in both cell types. Comprehensive chromatin immunoprecipitation analysis of the entire PSA locus and an androgen-response element in hMAK unexpectedly revealed that androgen receptor was not occupying any site in the absence of dihydrotestosterone in either cell type. In line with the expression data, and in the absence of dihydrotestosterone, histone acetylation and RNA polymerase II occupancy was substantial at the PSA locus in C4-2B but not in LNCaP cells. In the presence of dihydrotestosterone, androgen receptor was found to occupy mainly the enhancer region of PSA in both cell types, accompanied with increases in histone acetylation and RNA polymerase II occupancy. Although the androgen receptor was not directly involved in the androgen-independent expression of PSA in C4-2B cells, small interfering RNA knock-down of androgen receptor significantly reduced PSA expression in both the presence and absence of dihydrotestosterone. In contrast, hMAK expression was decreased only in the presence of dihydrotestosterone after androgen receptor knock-down. We conclude that androgen-independent expression of PSA in C4-2B cells does not rely on the direct occupancy of the androgen receptor at the PSA locus, but is nevertheless affected indirectly via unknown androgen receptor-dependent mechanism(s) that influence the expression from some but not all androgen receptor target genes.

Quantitative Analysis of Protein Expression Changes Induced by Geldanamycin in NPM-ALK Positive Lymphoma Cells by Functional Proteomics.

A subset of anaplastic large cell lymphomas are characterized by the t(2;5)(p23;q35) chromosomal translocation. Fusion of the nucleophosmin (NPM) gene on chromosome 5q35 with the anaplastic lymphoma kinase (ALK) gene on chromosome 2p23 results in the constitutive expression of the NPM-ALK oncoprotein. Heat shock protein 90 (HSP90) is a molecular chaperone involved in the folding of a number of substrate proteins implicated in cancer. The list of known HSP90 client proteins is expanding and includes oncogenes, cell cycle-associated proteins, and kinases including NPM-ALK. The ansamycin-based anti-cancer drug, geldanamycin (GA) has been shown to inhibit HSP90 by interfering with its function as a molecular chaperone, resulting in ubiquitin-mediated proteasomal degradation of its client proteins. To date, the cellular effects of inhibition of HSP90 by GA remain largely uncharacterized. Here, we describe a functional proteomics approach to identify the differentially expressed proteins in NPM-ALK positive lymphoma cells exposed to GA. GA induced a dose- and time-dependent decrease in cell viability with an IC50 of 20μM, which was associated with a G2/M cell cycle arrest followed by caspase-3 mediated apoptosis. Equal amounts of total cell lysates from DMSO-control and GA-treated SUDHL-1 cells were labeled with either the light or heavy cleavable ICAT™ reagent. Samples were combined, digested with trypsin, and separated by strong cation exchange chromatography and avidin affinity chromatography. Peptides were subjected to liquid chromatography (LC), electrospray ionization (ESI) and tandem mass spectrometry (MS/MS). The resulting peptides were searched against the NCBI and Swiss-Prot databases using SEQUEST™ for protein identification and relative quantitation determined by XPRESS™. A total of 117 proteins were differentially expressed by greater than 1.5-fold of which 48 were overexpressed and 69 were underexpressed in GA-treated cells relative to control. The 48 overexpressed proteins included those that are involved in apoptosis, protein degradation, cell communication, and cell cycle regulation. Of the 69 underexpressed proteins, many included proteins involved in ubiquitin-proteasome function (proteasome beta 2 subunit, HSP90-organizing protein, CBL-C), cytokines (IL-14 precursor), and DNA repair. Notably, a number of previously reported targets of HSP90/70 (male germ cell-associated kinase and IL-26 precursor) were identified. Our results provide insights into the role of HSP90 in the pathogenesis of NPM-ALK positive lymphomas and demonstrate the diverse cellular functions that are deregulated as a consequence of HSP90 inhibition.

Identification of Human Male Germ Cell-associated Kinase, a Kinase Transcriptionally Activated by Androgen in Prostate Cancer Cells

Androgen is involved in both normal development and malignant transformation of prostate cells. The signal transduction pathways associated with these processes are not well understood. Using a novel kinase display approach, we have identified a protein kinase, human male germ cell-associated kinase (hMAK), which is transcriptionally induced by the androgenic hormone 5alpha-dihydrotestosterone (DHT). The kinetics of induction is rapid and dose-dependent, and the induction is not blocked by cycloheximide treatment. Real time reverse transcription-PCR studies demonstrated a 9-fold induction of hMAK by 10 nm DHT at 24 h post-stimulation. The expression levels of hMAK in prostate cancer cell lines are in general higher than those of normal prostate epithelial cells. A reverse transcription-PCR product encompassing the entire hMAK open reading frame was isolated. The results from sequencing analysis showed that the hMAK protein is 623 amino acids in length and contains a kinase catalytic domain at its N terminus, followed by a proline/glutamine-rich domain. The catalytic domain of this kinase contains sequence motifs related to both the cyclin-dependent kinase and the mitogen-activated protein kinase families. When expressed in COS1 cells, hMAK is kinase-active as demonstrated by autophosphorylation and phosphorylation of exogenous substrate and is localized in the nucleus. A 3.7-kilobase pair promoter of the hMAK locus was isolated from a human genomic DNA bacterial artificial chromosome clone and was shown to be activated by DHT. This activation can be blocked by an anti-androgen drug bicalutamide (Casodex), implicating the involvement of androgen receptor in this process. Taken together, these data suggest that hMAK is a protein kinase targeted by androgen that may participate in androgen-mediated signaling in prostate cancer cells.

Specific Association of a Set of Molecular Chaperones Including HSP90 and Cdc37 with MOK, a Member of the Mitogen-activated Protein Kinase Superfamily

We have recently identified and cloned a novel member of mitogen-activated protein kinase superfamily protein, MOK (Miyata, Y., Akashi, M., and Nishida, E. (1999) Genes Cells 4, 299-309). To address its regulatory mechanisms, we searched for cellular proteins that specifically associate with MOK by co-immunoprecipitation experiments. Several cellular proteins including a major 90-kDa molecular chaperone HSP90 were found associated with MOK. Treatment of cells with geldanamycin, an HSP90-specific inhibitor, rapidly decreased the protein level of MOK, and the decrease was attributed to enhanced degradation of MOK through proteasome-dependent pathways. Our data suggest that the association with HSP90 may regulate intracellular protein stability and solubility of MOK. Experiments with a series of deletion mutants of MOK indicated that the region encompassing the protein kinase catalytic subdomains I-IV is required for HSP90 binding. Closely related protein kinases (male germ cell-associated kinase and male germ cell-associated kinase-related kinase) were also found to associate with HSP90, whereas conventional mitogen-activated protein kinases (extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase/stress-activated protein kinase) were not associated with HSP90. In addition, we found that other molecular chaperones including Cdc37, HSC70, HSP70, and HSP60 but not GRP94, FKBP52, or Hop were detected specifically in the MOK-HSP90 immunocomplexes. These results taken together suggest a role of a specific set of molecular chaperones in the stability of signal-transducing protein kinases.

A novel mammalian kinase, T/STK 30, is highly expressed in the germ line

In order to identify potential regulators of germ cell development in mammals we isolated serine/threonine kinases from adult testis because kinases have been found to play important roles in regulating both mitotic and meiotic cell cycles in a variety of organisms. One of the isolated genes, termed testis-derived serine/threonine kinase 30 (T/STK 30) revealed homology to the rat male germ cell associated kinase (Mak) and to a key regulator of entry into meiosis, the SME1 kinase from Saccharomyces cerevisiae (S. cerevisiae). T/STK 30 is most abundantly expressed in the testis and ovary and at low levels in the brain, but not in other adult tissues, during embryogenesis or in proliferating somatic cells. T/STK 30 transcripts were not detected in RNA from a sterile Steel (Sl) mutant testis, further demonstrating that T/STK 30 expression in the testis is limited to germ cells. Analysis of T/STK 30 expression in the developing testis revealed that T/STK 30 transcripts are detected in RNA from testis 17 days postpartum, but not 7 days postpartum, suggesting that T/STK30 expression is restricted to meiotic and postmeiotic germ cells. In situ hybridization analysis of T/STK 30 expression in the testis and ovary confirmed that this gene is highly expressed in germ cells. Thus T/STK 30 may be an important regulator of meiosis or postmeiotic differentiation in mammals. Mol. Reprod. Dev. 52:9–17, 1999. © 1999 Wiley-Liss, Inc.

Mouse Mak gene for male germ cell-associated kinase maps to chromosome 13.

Mouse Mak gene for male germ cell-associated kinase maps to chromosome 13.

Retinol differentially regulates male germ cell-associated kinase (mak) messenger ribonucleic acid expression during spermatogenesis.

Retinol down-regulates male germ cell-associated kinase (mak) transcripts during the spermatogonial proliferation phase of spermatogenesis. Mak transcripts of 2.6- and 3.4 kb were detected in vitamin A-deficient germ cells, but decreased to undetectable levels shortly after retinol replacement to vitamin A-deficient rats. In contrast, mak seems to play a role in spermatocytes and round spermatids during meiotic and post-meiotic events. The 3.4-kb transcript initially appeared in pachytene spermatocytes and continued to increase in round spermatids; the level of the 2.6-kb transcript increased markedly only in round spermatids. These transcripts also exhibited stage-specificity in synchronized retinol-regenerated rat seminiferous tubules. Silver grains were intensely localized mainly in round spermatids in stages IV-VI of the spermatogenic cycle in normal tests after in situ hybridization. These results suggest that the mak gene in early germ cells responds to retinol in an opposite manner from the mak gene in spermatocytes or in round spermatids. The findings support the existence of two distinct pathways of retinol signaling in the testis that depend on the spatial localization and development ages of the individual germ cells.

In situ localization of male germ cell-associated kinase (mak) mRNA in adult mouse testis: specific expression in germ cells at stages around meiotic cell division.

Biochemical analysis of the male germ cell-associated kinase (mak) gene, which was isolated recently by using weak cross-hybridization with the v-ros tyrosine kinase gene, revealed that the gene was highly expressed in mammalian testicular germ cells, but not in ovarian cells. In order to identify the cells which express the mak gene in more detail, we localized mak mRNA in frozen sections of mouse testis by non-radioactive in situ hybridization. In this study, we utilized thymine-thymine (T-T) dimerized mak cDNA as a haptenic, non-radioactive probe, and the signal was detected enzyme-immunohistochemically by using an anti-T-T antibody. As a result, mak mRNA was localized intensely in late pachytene (stage X) and diplotene (stage XI) spermatocytes, and faintly in dividing spermatocytes (stage XII) and early round spermatids (stage I-II), suggesting that the gene may play an important role in the phase around meiotic cell division, but not throughout the entire meiosis.

A Novel Mammalian Protein Kinase Gene (mak) Is Highly Expressed in Testicular Germ Cells at and after Meiosis

We isolated a novel gene designated mak (male germ cell-associated kinase) by using weak cross-hybridization with a tyrosine kinase gene (v-ros). Sequence analysis of the cDNA corresponding to the 2.6-kilobase transcript revealed that the predicted product of rat mak consisted of 622 amino acids and contained protein kinase consensus motifs in its amino-terminal region. Comparison of the deduced amino acid sequence of mak in the kinase domain with those of other protein kinase genes demonstrated that mak was approximately 40% identical to the cdc2-CDC28 gene family in Schizosaccharomyces pombe, Saccharomyces cerevisiae, and humans but less identical to most other protein kinase gene products. Expression of mak was highly tissue specific, and its transcripts were detected almost exclusively in testicular cells entering and after meiosis but hardly detectable in ovarian cells including oocytes, after the dictyotene stage. These results suggest that the mak gene plays an important role in spermatogenesis.

A novel mammalian protein kinase gene (mak) is highly expressed in testicular germ cells at and after meiosis.

We isolated a novel gene designated mak (male germ cell-associated kinase) by using weak cross-hybridization with a tyrosine kinase gene (v-ros). Sequence analysis of the cDNA corresponding to the 2.6-kilobase transcript revealed that the predicted product of rat mak consisted of 622 amino acids and contained protein kinase consensus motifs in its amino-terminal region. Comparison of the deduced amino acid sequence of mak in the kinase domain with those of other protein kinase genes demonstrated that mak was approximately 40% identical to the cdc2-CDC28 gene family in Schizosaccharomyces pombe, Saccharomyces cerevisiae, and humans but less identical to most other protein kinase gene products. Expression of mak was highly tissue specific, and its transcripts were detected almost exclusively in testicular cells entering and after meiosis but hardly detectable in ovarian cells including oocytes, after the dictyotene stage. These results suggest that the mak gene plays an important role in spermatogenesis.