Phosphorylation of Rat Spermatidal Protein TP2 by Sperm-specific Protein Kinase A and Modulation of Its Transport into the Haploid Nucleus

Abstract

Transition protein 2 (TP2), which is expressed during stages 12–15 of mammalian spermiogenesis, has been shown to undergo phosphorylation immediately after its synthesis. We reported earlier that TP2 is phosphorylated in vitro at threonine 101 and serine 109 by the salt extract of sonication-resistant (elongating and elongated) spermatid nuclei and the protein kinase phosphorylating TP2 was identified to be protein kinase A (PKA). We now report that the cytosol from haploid spermatids but not from premeiotic germ cells is able to phosphorylate recombinant TP2 in vitro at threonine 101 and serine 109. The kinase present in the haploid spermatid cytosol that phosphorylates TP2 has been identified to be the sperm-specific isoform of protein kinase A (Cs-PKA). Reverse transcription-PCR analysis indicated that Cs-PKA was present in the haploid spermatids and absent from premeiotic germ cells. The rat Cs-PKA transcript was amplified and sequenced using the isoform-specific primers. The sequence of rat Cs-PKA at the N terminus differs from mouse and human by one amino acid. Western blot analysis using specific anti-Cα1 antibodies revealed that Cα1-PKA is absent in haploid spermatid cytosol. We have also established an in vitro nuclear transport assay for the haploid round spermatids. Using this assay, we have found that the cytoplasmic factors and ATP are absolutely essential for translocation of TP2 into the nucleus. Phosphorylation was found to positively modulate the NLS dependent import of TP2 into the nucleus. Transition protein 2 (TP2), which is expressed during stages 12–15 of mammalian spermiogenesis, has been shown to undergo phosphorylation immediately after its synthesis. We reported earlier that TP2 is phosphorylated in vitro at threonine 101 and serine 109 by the salt extract of sonication-resistant (elongating and elongated) spermatid nuclei and the protein kinase phosphorylating TP2 was identified to be protein kinase A (PKA). We now report that the cytosol from haploid spermatids but not from premeiotic germ cells is able to phosphorylate recombinant TP2 in vitro at threonine 101 and serine 109. The kinase present in the haploid spermatid cytosol that phosphorylates TP2 has been identified to be the sperm-specific isoform of protein kinase A (Cs-PKA). Reverse transcription-PCR analysis indicated that Cs-PKA was present in the haploid spermatids and absent from premeiotic germ cells. The rat Cs-PKA transcript was amplified and sequenced using the isoform-specific primers. The sequence of rat Cs-PKA at the N terminus differs from mouse and human by one amino acid. Western blot analysis using specific anti-Cα1 antibodies revealed that Cα1-PKA is absent in haploid spermatid cytosol. We have also established an in vitro nuclear transport assay for the haploid round spermatids. Using this assay, we have found that the cytoplasmic factors and ATP are absolutely essential for translocation of TP2 into the nucleus. Phosphorylation was found to positively modulate the NLS dependent import of TP2 into the nucleus. The process of spermiogenesis in mammals, wherein the haploid round spermatids mature into highly condensed spermatozoa, can be broadly divided into three phases. In the first phase, encompassing stages 1–10, the round spermatids are transcriptionally active and contain nucleosomal chromatin. The second phase (stages 12–15) involves the replacement of nucleosomal histones by transition proteins TP1, TP2, and TP4. 1The abbreviations used are: TPtransition proteinPKAprotein kinase ACs-PKAspermatid-specific PKA isoformCα1-PKAsomatic PKA isoformFACSfluorescence-activated cell sortingGST-2KGST-2K, glutathione S-transferase with PKA recognition sitePKIcAMP-dependent protein kinase inhibitor peptideNLSnuclear localization signalSV40simian virus 40. Finally, in the third phase, the transition proteins are replaced by protamines P1 and P2 during stages 16–19 (1Meistrich M.L. Hnlica G. Stein G. Stein J. Histone and Basic Nuclear Protein Transitions in Mammalian Spermatogenesis. CRC, Orlando, FL.1989: 165-182Google Scholar, 2Wouters-Tyrou D. Martinage A. Chevaillier P. Sautiere P. Biochimie. (Paris). 1998; 80: 117-128Crossref PubMed Scopus (144) Google Scholar). It is quite interesting to note that the transformation of nucleohistone type of chromatin into nucleoprotamine fiber is not complete, and ∼10–15% of sperm chromatin retains nucleosomal histones, probably in the context of nucleosomal structure (3Uscheva A. Avramova Z. Tsanev R FEBS Lett. 1982; 138: 50-54Crossref PubMed Scopus (11) Google Scholar, 4Gatewood J.M. Cook G.R. Balhorn R. Bradbury E.M. Schmid C.W. Science. 1987; 36: 962-964Crossref Scopus (294) Google Scholar, 5Gatewood J.M. Cook G.R. Balhorn R. Schmid C.W. Bradbury E.M. J. Biol. Chem. 1990; 265: 20662-20666Abstract Full Text PDF PubMed Google Scholar, 6Pittoggi C. Renzi L. Zaccagnini G. Cimini D. Degrassi F. Giordano R. Magnano A.R. Lorenzini R. Lavia P. Spadafora C. J. Cell Sci. 1999; 112: 3537-3548PubMed Google Scholar, 7Meetei A.R. Ullas K.S. Rao M.R.S. J. Biol. Chem. 2000; 275: 38500-38507Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). These unexpected observations have raised several interesting questions as to the nature of chromatin domains, which resist this replacement process, and the possible significance of the chromatin domains that retain nucleosomal structure during early embryonic development. Over the last several years, we have been studying the DNA binding and condensing properties of the spermatid protein TP2 and have described in detail its molecular anatomy with respect to the nature and identity of novel zinc finger domains, the nuclear localization signal, and also the role of phosphorylation in DNA condensation (8Meetei A.R. Ullas K.S. Vasupradha V. Rao M.R.S. Biochemistry. 2002; 41: 185-195Crossref PubMed Scopus (35) Google Scholar, 9Yu Y.E. Zhang Y. Unni E. Shirley C.R. Deng J.M. Russell L.D. Weil M.M. Behringer R.R. Meistrich M.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4683-4688Crossref PubMed Scopus (237) Google Scholar). Based on our earlier observations, we have proposed a model describing the possible sequence of events from the time of TP2 synthesis in the cytoplasm and subsequent initiation of chromatin condensation upon deposition on chromatin, implicating distinct roles for the phosphorylation event and zinc finger domains (9Yu Y.E. Zhang Y. Unni E. Shirley C.R. Deng J.M. Russell L.D. Weil M.M. Behringer R.R. Meistrich M.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4683-4688Crossref PubMed Scopus (237) Google Scholar). According to this model, phosphorylation of TP2 at its C-terminal basic domain prevents the premature condensation of DNA, which allows the protein to scan the entire genome and bind to GC-rich sequences. Subsequent dephosphorylation at the basic domain leads to the initiation of chromatin condensation. transition protein protein kinase A spermatid-specific PKA isoform somatic PKA isoform fluorescence-activated cell sorting GST-2K, glutathione S-transferase with PKA recognition site cAMP-dependent protein kinase inhibitor peptide nuclear localization signal simian virus 40. Meistrich's group has generated knockout mice for both TP1 and TP2, and they have observed abnormal chromatin condensation in sperm isolated from the knockout mice (10Zhao M. Shirley C.R. Yu Y.E. Mohapatra B. Zhang Y. Unni E. Deng J.M. Arango N.A. Terry N.H. Weil M.M. Russell L.D. Behringer R.R. Meistrich M.L. Mol. Cell Biol. 2001; 21: 7243-7255Crossref PubMed Scopus (157) Google Scholar, 11Showers M.O. Maurer R.A. J. Biol. Chem. 1986; 261: 16288-16291Abstract Full Text PDF PubMed Google Scholar). More recently, they have also generated TP1–/–/TP2–/– double-knockout mice, which are infertile. 2M. L. Meistrich, personal communication. These phenotypes of the knockout mice stress the biological importance of the two proteins in mammalian spermatogenesis, particularly in the global chromatin remodeling process. In our previous study, we mapped, by site-directed mutagenesis, the major phosphorylation sites in TP2 to Thr-101 and Ser-109 in the C-terminal third of the protein, which shown to be phosphorylated by protein kinase A (8Meetei A.R. Ullas K.S. Vasupradha V. Rao M.R.S. Biochemistry. 2002; 41: 185-195Crossref PubMed Scopus (35) Google Scholar). These using the salt extract of the sonication-resistant spermatid and the of protein kinase A was by in vitro recombinant TP2 and its and protein kinase A. The PKA of two to two of somatic are which is expressed in of the M.O. Maurer R.A. J. Biol. Chem. 1986; 261: 16288-16291Abstract Full Text PDF PubMed Google Scholar, Proc. Natl. Acad. Sci. U. S. A. 1986; PubMed Scopus Google Scholar, J. Biol. Chem. 1986; 261: Full Text PDF PubMed Google expressed at in M.O. Maurer R.A. J. Biol. Chem. 1986; 261: 16288-16291Abstract Full Text PDF PubMed Google Scholar, J. Biol. Chem. 1986; 261: Full Text PDF PubMed Google and which is a and is expressed in M. A. V. Mol. 1990; PubMed Scopus Google Scholar, S. V. 1998; PubMed Scopus Google Scholar). More recently, a sperm-specific isoform of has been reported Mol. Biol. 2000; PubMed Scopus Google Scholar). G.R. Balhorn R. Mol. PubMed Scopus Google that the with the with TP2 was in the cell extract that with TP2 in sonication-resistant this to that TP2 phosphorylation immediately after its synthesis in the this in we to the nature of the protein kinase present in the round spermatid cytoplasm that is in TP2 In the present study, we this and that the sperm-specific isoform of the of protein kinase A is in the phosphorylation of the phosphorylation sites to be the nuclear localization signal of TP2, we also the of phosphorylation on its import into the and found that the transport is by phosphorylation but is not for its import into the nucleus. of round spermatids isolated from cells of by the of as we have described L. Rao M.R.S. J. Biol. Chem. 1990; 265: Full Text PDF PubMed Google Scholar). The isolated cells in and an was with and with to the by was from round spermatids and also from the cells of and by the cells in and in with inhibitor and by at for at The cytosol was by to a protein of and at In and In of proteins and of recombinant TP2 the PKA in a and zinc for at of was in the of peptide was with cytosol the of protein The was by the of and at for The on a with and to In kinase assay was as described M. Scholar, M. M.L. 1986; PubMed Scopus Google with of protein from round spermatids was on a with the recombinant the the was with and the proteins in and for a of with in a and The was in kinase for at and in the of with to the was and to proteins on a TP2 as a Western with PKA and of and Cs-PKA by from round spermatids and cells from and on a and a The proteins using antibodies raised an peptide to to was used as the with as the was isolated from round spermatids and cells of and using the Reverse was using the for both the and and the was using the isoform-specific for Cα1-PKA and for and the as described by Mol. Biol. 2000; PubMed Scopus Google Scholar). of rat was used as the and as the The DNA of the in an DNA In Nuclear transport of recombinant TP2 in vitro with cells was to the of L. J. Cell Biol. 1990; PubMed Scopus Google with the round spermatids after with transport 2 and inhibitor and The cells in transport at for and with transport of and TP2 used for in vitro transport In vitro transport was in transport TP2, and and with and The import was to for at In the indicated import was in transport in the of ATP Nuclear import assay in cytosol ATP was also as one of the the import the cells with transport on and the localization of TP2 was by using as described A.R. Ullas K.S. Rao M.R.S. J. Biol. Chem. 2000; 275: 38500-38507Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). the cytosol was with inhibitor peptide for PKA in nuclear import of TP2 and in TP2 was into at sites that TP2 was in at the terminus of cells with type the using to the by the cells on in a in with at in after the cells with and with proteins in an of of in cells on and round spermatids was by using antibodies the cells with with with and with with was used as the Phosphorylation of TP2 by have shown that rat TP2 is phosphorylated in by with and in vitro by the protein kinase A present in the salt extract of sonication-resistant spermatid nuclei (8Meetei A.R. Ullas K.S. Vasupradha V. Rao M.R.S. Biochemistry. 2002; 41: 185-195Crossref PubMed Scopus (35) Google Scholar). The PKA phosphorylation sites to the C-terminal third of the and identified to be Thr-101 and which Ser-109 to be the site spermatid nuclei a of both (stages 12–15) and (stages spermatids. earlier the two major basic nuclear proteins present in the spermatids (stages 12–15) are TP1 and TP2 and in the spermatids (stages are protamines P1 and the nuclei from which the salt extract was contain TP2 to chromatin, we to the phosphorylation of TP2 in the cytosol of round particularly TP2 is to undergo phosphorylation immediately after its synthesis G.R. Balhorn R. Mol. PubMed Scopus Google Scholar). this we have isolated the haploid round spermatids from cells by the of analysis of the isolated round spermatids is shown in the was by cells. also the of and cells in the cells from which round spermatids The of in vitro kinase with recombinant TP2 as the and cytosol from round spermatids and cells from and are shown in The germ cells in the of premeiotic and a early from contain a of and premeiotic germ cells. haploid round spermatids in the of at can be from TP2 was phosphorylated by the round spermatid cytosol but not by the cytosol of rat cells and The also that the and of TP2 is not phosphorylated by the round spermatid cytosol. is that the round spermatid cytosol phosphorylated the serine and threonine that we observed earlier with the salt extract of sonication-resistant spermatid The of phosphorylation of TP2 by the cytosol of rat cells was in our assay and also to cytosol contain we using PKA as the of The in that is phosphorylated by the cytosol that PKA is present in the of and cells but not phosphorylate that PKA is phosphorylating TP2 in the round spermatid we phosphorylation assay in the of of a specific inhibitor of can be in was a of phosphorylation of TP2 with of that PKA present in round spermatid cytosol is phosphorylating the We also kinase assay to the molecular of the kinase present in the round spermatid cytosol that is phosphorylating The of an in a of which with the molecular of PKA of a probably of the PKA of round spermatids and in vitro kinase of cells from of round spermatids from haploid cells. in vitro kinase assay of recombinant TP2 was as described and the on and to The kinase with from cells cells and round spermatids and is the TP2 TP2 used as of the type recombinant TP2 in vitro kinase assay of was using the from rat cells rat cells and round spermatids and is the TP2 and in and TP2, an at GST-2K, of in vitro kinase assay by PKA inhibitor peptide and kinase in vitro kinase assay of recombinant type TP2 was as described and the on and to kinase with cytosol from round spermatids in the of of peptide inhibitor of the of the assay in the of the inhibitor the of recombinant type kinase assay was as using round spermatid cytosol as the of kinase and TP2 in the as the The a which in the the molecular of the kinase that phosphorylates protein molecular of PKA in group Mol. Biol. 2000; PubMed Scopus Google Scholar, Biol. 2001; PubMed Scopus Google has identified and the of the sperm-specific isoform of the Cs-PKA in mouse and Cs-PKA have a and during in the PKA is expressed in somatic and The report by Biol. 2001; PubMed Scopus Google that from the cells with the of analysis using Cs-PKA specific antibodies revealed that the of from Cs-PKA has also been shown to be present in the cytoplasm of round and spermatids Biol. 2001; PubMed Scopus Google Scholar). have also shown that is absent in somatic cells and germ and early Cs-PKA has also been in human and These two from in amino sequence antibodies are peptide to which not with we to the nature of PKA present in the cytosol of rat round spermatids. A and the of Western blot analysis in which is that the PKA specific antibodies not the PKA present in the round spermatid but with the Cα1-PKA isoform present in the cytosol of and cells. We the of Cα1-PKA and Cs-PKA isoform by analysis using isoform-specific at the of These are shown in and D. The transcript to Cα1-PKA isoform was in the cells of and but not in round spermatids of the the Cs-PKA isoform transcript was in round spermatids. the of and Cs-PKA in rat germ cells that has also been reported in the of and human Mol. Biol. 2000; PubMed Scopus Google Scholar, Biol. 2001; PubMed Scopus Google Scholar). also that Cs-PKA is for TP2 phosphorylation in round spermatid cytosol. The and Cs-PKA in mouse also in the type of at N in the of Cs-PKA and of the we observed in kinase assay A that can be in the present is that the recombinant TP2 was not phosphorylated by the of and rat cells they contain and analysis of and Western blot analysis and analysis to the kinase in the phosphorylation of TP2 and to the of and Cs-PKA of the using PKA rat rat round spermatid molecular and with specific and a with and a and the from rat rat and round spermatids molecular We have also amplified the entire of the rat Cs-PKA isoform and its which has been to The sequence at the and the amino sequence of rat Cs-PKA isoform are shown in with the of and human Cs-PKA and the somatic isoform In of the first amino of the N terminus of are replaced by a in the Cs-PKA It is interesting to note that is a in the Cs-PKA isoform of at in a of one amino Mol. Biol. 2000; PubMed Scopus Google Scholar, Biol. 2001; PubMed Scopus Google observed that antibodies the Cs-PKA isoform of mouse to not with the Cs-PKA isoform of probably of this amino have also shown that the Cα1-PKA and Cs-PKA isoform are generated by the of sites Mol. Biol. 2000; PubMed Scopus Google and the two first are into the of the The sequence of rat Cs-PKA and Cs-PKA of and the first amino found to be to that of In of TP2 into and of of nuclear proteins an role in import from the particularly the of NLS G. U. Full Text PDF PubMed Scopus Google Scholar, Stein D. J. A. S. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, R. J. PubMed Scopus Google Scholar, R. J. Cell Biol. PubMed Scopus Google Scholar). We have identified that the NLS sequence of TP2 the and to the of that is present in phosphorylation by protein kinase Cs-PKA isoform on Thr-101 and which are immediately of the NLS we to the of on the import of TP2 into the round spermatid nucleus. are after of the in the cell we in a for round spermatids using the now for cell is also report in the describing a we to the of using nuclear import assay with round which have been used in the to the of nuclear import L. J. Cell Biol. 1990; PubMed Scopus Google Scholar). In we have the import of recombinant TP2 and its by The of are in the of the localization of recombinant round spermatids with transport in the and of the ATP the protein was in the of a the of the nuclear the transport assay was in the of TP2 was into the nuclear import of TP2 in the of cytosol is dependent on the of ATP These that transport of TP2 into the round spermatid is by cytoplasmic protein and is We also to to the nature of observed the round spermatid in our import assay We shown in that TP2 to in cells. to that localization to is not an isolated we have with cells and that in TP2 to the protein to in cells by have been to be in round spermatids and to during the phase transition proteins are expressed during of was using in cells and round spermatids can be from the in cells not observed in the round spermatid that the in the of TP2, the round spermatid are not and probably localization of TP2 to GC-rich the import assay with type TP2, we the assay with the of The of localization of TP2 is shown in observations can be from this the cytoplasmic of TP2 is quite from that of type TP2 observed in TP2, as in the of TP2, and of the protein was the nuclear as was observed with cytosol A second was that also of TP2 localization the as observed with the of TP2 in the is the We have also the import with TP2 in the of an inhibitor of protein kinase A. can be from of the an the nuclear which was to that of the phosphorylation site The cytoplasmic as as nuclear was with the the of two that phosphorylation positively the transport of TP2 into the round spermatid as in the of R. J. PubMed Scopus Google Scholar, R. J. Cell Biol. PubMed Scopus Google Scholar). We have also this import assay for and and have observed in the of TP2 and phosphorylation site TP2 not In the present study, we that rat protein TP2 is phosphorylated at Thr-101 and Ser-109 by the sperm-specific PKA present in the cytoplasm of haploid round spermatids. The of the somatic isoform and the sperm-specific isoform Cs-PKA during rat with the observed in mouse Mol. Biol. 2000; PubMed Scopus Google Scholar, Biol. 2001; PubMed Scopus Google Scholar). The of Cs-PKA has also been in the sperm and is to be for of is not in and is A in the present is that the Cα1-PKA present in the cytosol of and rat cells not phosphorylate recombinant be in the context of our previous that TP2 can be phosphorylated in vitro by PKA (8Meetei A.R. Ullas K.S. Vasupradha V. Rao M.R.S. Biochemistry. 2002; 41: 185-195Crossref PubMed Scopus (35) Google Scholar). It is quite that the of PKA as in as with which of In this sperm-specific proteins have also been reported in the J. J. PubMed Scopus Google Scholar, Biol. 1999; PubMed Scopus Google Scholar). We have also in the present study, an in vitro import assay for studying the of nuclear proteins into the haploid round spermatids. These that transport of TP2 into the round spermatid is by cytoplasmic protein and is It is now established that nuclear import of proteins is a process a of of which the the NLS and as the to L. Full Text PDF PubMed Scopus Google Scholar, D. F. E. R.A. PubMed Scopus Google Scholar). are to an NLS The nuclear and are the nuclear in an of in the from the D. Cell Biol. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). The of TP2 in the cytoplasm of round spermatids a the It to be TP2 a with with isoform to has been shown to be in the nuclear import and of binding as proteins and S. D. J. 1998; PubMed Scopus Google Scholar, S. U. D. D. J. 1999; PubMed Scopus Google Scholar, S. J.M. P. E. D. J. 2002; 21: PubMed Scopus Google Scholar). It is that proteins a NLS to be to the and proteins of molecular can into the nucleus. on import of S. U. D. D. J. 1999; PubMed Scopus Google Scholar, M. 1990; Full Text PDF PubMed Scopus Google Scholar, M. D. J. Cell PubMed Scopus Google and proteins S. D. J. 1998; PubMed Scopus Google Scholar, G. Full Text Full Text PDF PubMed Scopus Google Scholar, G. E. R. J. U. D. J. PubMed Scopus Google have indicated that proteins and that to be into the on specific they are for It has also been that nuclear import to the its have been for basic as histones which have a to and at salt S. U. D. D. J. 1999; PubMed Scopus Google Scholar). It is interesting to note that TP2 is a highly basic protein and has a molecular of but has a NLS and a of cytoplasmic for its import into the that is a process. can be that TP2 is an nuclear transport to that its and is not These questions are in a and be reported The of TP2 localization in round spermatids not the localization that we observed earlier in cells A.R. Ullas K.S. Rao M.R.S. J. Biol. Chem. 2000; 275: 38500-38507Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). We have now shown that the of localization was not an isolated with cells but was also observed in cells using both TP2 and and The of in round spermatids indicated the of active in cells of a localization in in round spermatid was and that haploid spermatids are in and that the are during the is now that haploid spermatids are active in 1998; PubMed Scopus Google Scholar). a of has not been the of in haploid spermatids to be are in a is the and structure in the of a is to be by of the and a of proteins D. P. J. J. Cell Sci. 2002; PubMed Google Scholar). In the of a the be the in round spermatid It be interesting to TP2 chromatin domains that are the round spermatid in import of In a to be reported we have using chromatin assay, that the TP2 DNA in spermatids (stages to are The import assay reported have also that the import of type TP2 in the of peptide the of TP2 is not as as that of type TP2, that the NLS of TP2 to the of proteins that are by a of sequence the of is by phosphorylation by protein kinase and the kinase R. J. PubMed Scopus Google Scholar, R. J. Cell Biol. PubMed Scopus Google Scholar). of site by the PKA site also to be by that this is not kinase specific S. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the of NLS an of of an NLS by phosphorylation is the kinase of G. U. Full Text PDF PubMed Scopus Google Scholar). It is quite that of transport by phosphorylation be the of the with the in the The of NLS in TP2 and the of the nuclear import by a phosphorylation event is to that observed in the of the phosphorylation of TP2 to have an role in to the chromatin as has been proposed in our model (8Meetei A.R. Ullas K.S. Vasupradha V. Rao M.R.S. Biochemistry. 2002; 41: 185-195Crossref PubMed Scopus (35) Google Scholar). In this model, we proposed that phosphorylation of the basic domain of TP2 its condensation to the zinc to and the in chromatin. A event has been proposed for deposition on sperm chromatin R. Mol. Biol. PubMed Scopus Google Scholar). model also that a be present in spermatid to TP2 and to chromatin condensation. In this is that S. A. J. Biol. 1999; PubMed Scopus Google have reported that the protein is expressed in round and mice for this are infertile.