Stage-specific Expression of Dynein Light Chain-1 and Its Interacting Kinase, p21-activated Kinase-1, in Rodent Testes : Implications in Spermiogenesis
Department of Molecular and Cellular Oncology (R-AW,RK) and Department of Experimental Radiation Oncology (MZ,MLM), UT-M.D. Anderson Cancer Center, Houston, Texas
Correspondence to: Rakesh Kumar, PhD, Department of Molecular and Cellular Oncology, Box 108, UT-M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. E-mail: rkumar{at}mdanderson.org
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Summary |
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Key Words: DLC1 Pak1 testis spermatogenesis
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Introduction |
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Cytoplasmic dynein is a large, multisubunit molecular motor complex that translocates cargoes toward the minus ends of microtubules. The dynein complex was originally shown to regulate the movement of chromosomes, the assembly and orientation of mitotic spindles, and nuclear migration (Steuer et al. 1990; Vaisberg et al. 1993
; Holzbaur and Vallee 1994
; Beckwith et al. 1998
; King 2000
). Dynein light chain 1 (DLC1), a subunit of the dynein motor complex, is a highly conserved small protein composed of 89 amino acids. In addition to playing an essential role in the dynein motor complex, DLC1 also regulates other biological events by binding to various proteins and enzymes. For example, DLC1 binds to and inhibits neuronal nitric oxide synthase and thus is also known as a protein inhibitor of neuronal nitric oxide synthase (Jaffrey and Snyder 1996
). DLC1 is also known to bind to other proteins, such as the proapoptotic Bcl-2 family member protein, Bim (Puthalakath et al. 1999
); the Drosophila RNA localization protein, Swallow (Schnorrer et al. 2000
); myosin V (Puthalakath et al. 2001
); neuronal scaffolding protein, GKAP; and I
B
, an inhibitor of the NF
B transcription factor (Naisbitt et al. 2000
). Despite the widespread expression of DLC1, its specific functions in different tissues and organs remains unknown, with the exception of its role in Drosophila. In Drosophila, partial loss of function of DLC1 leads to morphogenetic defects, female sterility, alterations in axonal guidance, and total loss of function, resulting in apoptosis and embryonic death (Dick et al. 1996
; Phillis et al. 1996
). In addition, we have recently shown that p21-activated kinase 1 (Pak1), a serine/threonine kinase, interacts with and phosphorylates DLC1 at Serine 88, and that Pak1-DLC1 interactions play a fundamental role in cell survival (Vadlamudi et al. 2004
).
Protein kinases, the enzymes responsible for the phosphorylation of a wide variety of proteins, are the largest class of signaling molecules known to regulate growth, development, and neoplastic transformation (Kumar and Wang 2002). Pak1 was originally discovered as a cytoskeleton-associated protein kinase. Pak1 is involved in the maintenance of cell shape, cell migration, and cell survival (Kumar and Vadlamudi 2002
). In addition, Pak1 has also been localized in the cell nucleus, where it appears to be involved in the phosphorylation of histones and transcription factors, such as estrogen receptor and Stat5a (Li et al. 2002
; Wang et al. 2002
,2003
). In the present report, we document that DLC1 and Pak1 are highly expressed in mouse and rat testes in a developmentally regulated, stage-specific manner, and that Pak1 regulation of the testis-specific isoform of cAMP-responsive element modulator (CREM
) might play an important role in the regulation of DLC1 expression in testes.
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Materials and Methods |
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RT-PCR and Northern and Western Blotting
For RT-PCR, we used the Access RT-PCR system (Promega Corp.; Madison, WI) and followed the protocols suggested by the manufacturer. The primers used were F-CTGTCTTCTGCTGCTTGAGCG and R-GTACACAACACAACAGATTCAAACGAG. For Northern blot analysis, 20 µg of total RNA was resolved on a 1% formaldehyde agarose gel, transferred to a nylon membrane, probed with appropriate probes, and exposed to a phosphoscreen. For immunoblot analysis, 40 µg of total protein lysate was resolved on a 15% SDSpolyacrylamide gel, transferred to a nitrocellulose membrane, and probed with appropriate antibodies.
Immunohistochemistry and Immunofluorescence Studies
Immunohistochemical staining was done with the indirect enzyme labeling method, as described previously (Wang and Zhao 1999). Briefly, the sections were dewaxed in xylene and rehydrated by exposure to descending concentrations of ethanol, immersed in 0.3% H2O2/methanol for 30 min, and washed with PBS. The sections were then sequentially incubated with 1% normal rabbit serum for 1 hr; mouse anti-DLC1 (1:200; Transduction Lab, Cat # 610,726), rabbit anti-CREM
(1:250, a gift from Dr. Paolo Sassone-Corsi, France), or rabbit anti-Pak1 (1:50; Santa Cruz Biotechnology, Santa Cruz, CA) for 2 hr; then with horseradish peroxidaseconjugated donkey anti-mouse or anti-rabbit IgG (1:100; Amersham Biosciences, Piscataway, NJ) for 1 hr, with PBS washes after each incubation. Finally, the antigen sites were visualized with diaminobenzidine/H2O2 solution, and the sections were counterstained with Mayer's hematoxylin for 30 sec. For specificity control, the primary antibody was replaced with normal mouse or normal rabbit serum. In the case of immunofluorescence studies, the second antibody was replaced with FITC-labeled anti-mouse IgG (1:200; Molecular Probes).
Plasmids
Pak1 expression plasmids have been described previously (Adam et al. 1998). pSV-CREM was provided by Dr. Sassone-Corsi. DLC1 promoter luciferase constructs were made by amplifying the upstream fragments of DLC1 from the BAC clone and put into the pGL3-basic vector at the site of KpnI/XhoI.
Cell Culture, Transfection, and Luciferase Assay
COS-7 cells were maintained in DMEM/F12 medium supplemented with 5% fetal calf serum. Cells were plated in six-well culture plates, and transfection was performed using the Fugene (Roche Diagnostics; Basel, Switzerland) method. Luciferase assay was performed according to the manufacturer's instructions, as described previously (Mazumdar et al. 2001), and the results were standardized against the ß-gal luciferase activity shown by the internal control. Each experimental group included three triplicate plates.
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Results |
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Stage-specific Expression of DLC1
We next conducted immunohistochemical analysis to further elucidate the expression pattern of DLC1 in testes. Although Western blotting showed more DLC1 in testes than in other tissues before active spermiogenesis, this was not confirmed by immunohistochemistry. This was probably because of the different detection limits of the two methods. Nevertheless, we found a very strong DLC1 immunoreactivity in the luminal side of the multilayered seminiferous tubules soon after the onset of active spermiogenesis at the age of 30 days (Figure 3).
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To test this hypothesis, we first evaluated the expression of CREM in mouse testes. As previously reported (De Cesare et al. 1999
), the expression of CREM in mouse testes was confined to the nucleus of round spermatids, especially in a high level at step 7 (Figure 7), during which high level of Pak1was also detected (Figure 6D). Next, we cloned the DLC1 promoter from BAC clones into the pGL3 luciferase vector. Sequence analysis done with TransFac software (Biobase; Braunschweig, Germany) revealed that there were two putative CREB binding sites on the DLC1 promoter (Figure 8A). We next examined the effect of CREM and Pak1 on DLC1 promoter activity using a COS-7 cell cotransfection assay. This showed a significant increase in DLC1 promoter activity when Pak1 and CREM were cotransfected, but neither Pak1 nor CREM alone affected DLC1 promoter activity (Figure 8B).
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Discussion |
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Given that the testis is a highly dynamic organ in which cells are constantly proliferating and differentiating, it is very likely that dynein complex components are involved in these processes. Indeed, two other components of the dynein light chain, Tctex1 and Tctex2, have also been documented to be expressed in testes and found to be a component of sperm flagella. Tctex1 and Tctex2 are believed to be involved in the non-Mendelian transmission of the t-haplotype in mice (Criswell and Asai 1998; Harrison et al. 1998
). Though not fully understood, transmission ratio distortion (also called meiotic drive) is believed to be related to abnormal spermiogenesis (Harrison et al. 1998
). The resultant abnormally developed sperm have little capacity to fertilize ova. Our study provided detailed documentation of DLC1 expression in the spermatids, which has several important functional implications and sheds some light on the mechanism that can go awry in spermiogenesis: first, DLC1 is not only a cytoplasmic protein, but it may also function in the nucleus, as has been recently shown in cultured cells using subcellular fractionation methods (Kaiser et al. 2003
). In our study, we postulated that DLC1 plays an important motor function during early-stage chromatin condensation. Second, after initial chromatin condensation, the spermatid needs to streamline its shape and get rid of excess cytoplasm. All of these processes require active movement of the microtubule-associated cytoskeletons. In addition, DLC1 may also play a role in reshaping spermatid. Third, DLC1 may provide an important motor drive for the final release of mature sperm into the lumen. The coexpression of Pak1 and DLC1 in elongated spermatid stage suggests that DLC1 maybe activated through phosphorylation by Pak1.
A time lag between transcription and translation is common in testes. It is reasonable to speculate that the transcription of these genes that encode proteins expressed in elongated spermatids must be inactivated in these spermatids, and, therefore, the coding mRNAs must be completed earlier. In addition to DLC1, protamines and transitional proteins are other good examples of tightly controlled protein expression (Hecht 1998). The pretranscribed mRNA remains free, without polysomal loading. The repression of translation of these mRNAs is likely to be related to RNA binding proteins that recognize the 3'-UTR region of the transcripts (Hecht 1998
). The repression of earlier translation is important for spermiogenesis. In the case of protamine, when the 3'-UTR was modified and protamine protein was expressed in the round spermatids, the mouse became infertile because of the structural changes in the sperm chromatin (Braun et al. 1989
). The time lapse between DLC1 mRNA transcription and protein expression also raises the possibility that DLC1 is important in chromatin condensation and the final morphogenesis of sperm.
In summary, we found that DLC1 is highly expressed in elongated spermatids, initially in the nucleus and subsequently in the cytoplasm. The stage-specific expression of DLC1 in elongated spermatids suggests that DLC1 may be involved in chromatin condensation, morphologic changes in nucleus, the shearing off of excess cytoplasm, and release of sperm into the lumen.
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Acknowledgments |
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We thank Dr. Paolo Sassone-Corsi for kindly providing us with the CREM expression vector and antibody, and David Galloway for editing of the manuscript.
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Footnotes |
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Literature Cited |
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