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Originally published In Press as doi:10.1074/jbc.M311949200 on December 30, 2003

J. Biol. Chem., Vol. 279, Issue 12, 11626-11631, March 19, 2004
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Temporal Association of Protamine 1 with the Inner Nuclear Membrane Protein Lamin B Receptor during Spermiogenesis*

Ilias Mylonis{ddagger}, Victoria Drosou{ddagger}, Stefano Brancorsini§, Eleni Nikolakaki{ddagger}, Paolo Sassone-Corsi§, and Thomas Giannakouros{ddagger}

From the {ddagger}Laboratory of Biochemistry, Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki 54 124, Greece and the §Institut de Génétique et de Biologie Moléculaire et Cellulaire, B.P. 10142, 67404 Illkirch, Strasbourg, France

Received for publication, October 31, 2003 , and in revised form, December 22, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During mammalian spermiogenesis, histones are replaced by transition proteins, which are in turn replaced by protamines P1 and P2. P1 protamine contains a short arginine/serine-rich (RS) domain that is highly phosphorylated before being deposited into sperm chromatin and almost completely dephosphorylated during sperm maturation. We now demonstrate that, in elongating spermatids, this phosphorylation is required for the temporal association of P1 protamine with lamin B receptor (LBR), an inner nuclear membrane protein that also possesses a stretch of RS dipeptides at its nucleoplasmic NH2-terminal domain. Previous studies have shown that the cellular protein p32 also binds tightly to the unmodified RS domain of LBR. Extending those findings, we now present evidence that p32 prevents phosphorylation of LBR and furthermore that dissociation of this protein precedes P1 protamine association. Our data suggest that docking of protamine 1 to the nuclear envelope is an important intermediate step in spermiogenesis and reveal a novel role for SR protein kinases and p32.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The development of spermatids into spermatozoa, termed spermiogenesis, is characterized by the replacement of histones by the highly basic, arginine-rich, protamines (1). As a result of this exchange, the nucleosomal-type chromatin is transformed into a smooth fiber and compacted in a volume of about 5% of that of a somatic cell nucleus (2, 3). Although the exchange of chromatin proteins during spermiogenesis has long been known, the molecular mechanisms and the signaling pathways governing the histone to protamine transition have remained obscure.

The deposition of protamines on sperm chromatin and the subsequent chromatin condensation appear to be controlled by phosphorylation-dephosphorylation events. Protamines are highly phosphorylated, shortly after their synthesis and before binding to DNA, whereas they become largely dephosphorylated during sperm maturation (4–8). Phosphorylation of P2 protamine has been shown to be essential, because deletion of the calmodulin-dependent protein kinase Camk4, which phosphorylates P2 protamine, impairs the replacement of transition protein-2 with P2 protamine, resulting in defective spermiogenesis and male sterility (9). On the other hand, all P1 protamines contain short arginine/serine-rich (RS)1 domains that are efficiently phosphorylated by SRPK1 (SR protein kinase 1) (10), but the physiological significance of this modification is mostly unknown.

In this respect, Biggiogera et al. (11) reported that protamines initially appear at the nuclear periphery, implying that the nuclear envelope might play a role in the replacement of transition proteins by protamines during spermiogenesis. Given that RS domains mediate protein-protein interactions (12), we sought to investigate the potential interaction of P1 protamine with the inner nuclear membrane protein lamin B receptor (LBR), which also possesses a repeat of RS dipeptides at its nucleoplasmic NH2-terminal domain. In the present study we demonstrate a direct association of these two proteins, in a phosphorylation-dependent manner.

SRPK1 was initially purified and cloned on the basis of its ability to phosphorylate and regulate the intracellular localization of SR splicing factors that commit precursor mRNA to splicing and promote spliceosome assembly (13). A polypeptide with an apparent molecular mass of 32 kDa (p32), isolated first as a protein tightly associated with the essential SR splicing factor ASF/SF2 (14), was shown to inactivate ASF/SF2 as both a splicing repressor and splicing enhancer protein (15). p32 was also found to form an in vivo complex with LBR (16, 17). In this study we reinvestigated the significance of the p32-LBR interaction, presenting evidence that p32 prevents LBR phosphorylation and P1 protamine association with LBR. Immunofluorescence analysis suggests that dissociation of this protein precedes P1 protamine association in early spermiogenesis. Taken together our data provide some insights into the molecular machinery that spatially directs the histone to protamine transition, revealing a new role for SR protein kinases and p32.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids, Expression of Proteins, and Antibodies—Human SRPK1, the wild-type NH2-terminal domain of LBR (GST-wtNt; amino acids 1-205), and a mutant form lacking the RS motifs (GST-{Delta}RS; lacks amino acids 75–84; 75RSRSRSRSRS84) were expressed as fusion proteins with GST using the pGEX-2T vector (Amersham Biosciences) (18, 19). Full-length SRPK1 was also subcloned into the p-FLAG-CMV-2 (Eastman Kodak) vector and expressed in 293T cells with a FLAG tag fused at its NH2 terminus (20). 293T cells were transiently transfected with 3–5 µg of plasmid DNA using the CaCl2 method and harvested 48 h later (20). The purified, nonphosphorylated form of human protamine P1 was a generous gift from Philippe Chevaillier (Laboratoire de Biologie Cellulaire, Université Paris-Val de Marne, 94010 Créteil, France). Recombinant p32 (kindly provided by G. Akusjärvi, Department of Medical Biochemistry and Microbiology, Uppsala University, S-75123 Uppsala, Sweden) was isolated as a His-tagged protein by standard nickel column chromatography (Qiagen) according to the manufacturer's instructions. The polyclonal anti-protamine 1 antibody was generated by immunizing rabbits with a keyhole limpet hemocyanin-coupled peptide (MARYRCCRSKSRSRCR) corresponding to the NH2-terminal sequence (residues 1–16) of mouse protamine 1. A rabbit anti-LBR polyclonal antibody raised against GST-wtNt was kindly provided by P. B. Singh (Division of Gene Expression and Development, The Roslin Institute, Edinburgh, UK), whereas the M5 anti-FLAG monoclonal antibody was a kind gift from G. Mosialos (Biomedical Sciences Research Center "Al. Fleming," Vari, Attiki). The polyclonal antibodies against p32 and SRPK1 were previously characterized (17, 20).

Preparation of Testis Cytosolic and Nuclear Extracts—Testes from 5-month-old Wistar rats or C57 BL/6 mice were homogenized by means of a Potter-Elvehjem tissue homogenizer in 3 volumes of an ice-cold solution containing 0.25 M sucrose, 50 mM Tris-HCl, pH 7.5, 25 mM KCl, 5 mM MgCl2, 5 mM dithiothreitol, and 3 mM phenylmethylsulfonyl fluoride. The homogenate was passed through two layers of surgical gauze and then centrifuged at 1000 x g for 10 min at 4 °C to yield a crude nuclear fraction. The supernatant was centrifuged at 100,000 x g for 2 h to obtain the cytosolic fraction (S100). The nuclei were further purified from the crude nuclear fraction by centrifugation through a cushion of 2.3 M sucrose at 100,000 x g for 90 min according to the method of Blobel and Potter (21). The nuclear pellets were resuspended in 10 mM Na2HPO4/NaH2PO4, pH 7.5, 2 mM MgCl2, and 1 mM phenylmethylsulfonyl fluoride and digested with 40 µg/ml DNase I for 30 min at room temperature. NaCl to a final concentration of 1 M was then added, and the mixture was further incubated at 4 °C for 2 h on a rotator. Nuclear extracts were collected by centrifugation at 10,000 x g for 30 min at 4 °C. Protein concentration was determined by the method of Bradford (22).

Kinase Assays—In vitro phosphorylation assays were carried out at 30 °C in a total volume of 25 µl containing 25 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 50 mM NaCl, 50 µM [{gamma}-32P]ATP (6000 Ci/mmol), 2–3 µg of the appropriate substrate (bacterially expressed LBR or P1 protamine), and an aliquot of the enzyme as indicated. The samples were incubated for 30 min, and the reaction was stopped by adding 6 µl of 5x SDS sample buffer and heating at 95 °C for 3 min. In situ kinase assays using rat testis cytosolic and nuclear extracts were performed as previously described (18), following essentially the protocol of Kameshita and Fujisawa (23). GST-wtNt was added to the separating gel at a concentration of 0.1 mg/ml prior to polymerization. For control experiments GST-wtNt was replaced by 0.1 mg/ml GST.

GST Pull-down Experiments—Incubation of GST, GST-wtNt, or GST-{Delta}RS (2–3 µg each) immobilized on glutathione-Sepharose beads with purified P1 protamine (~3 µg) or mouse testis nuclear extracts (~300 µg of total protein) was performed in TNMT buffer (20 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 2 mM MgCl2, and 1% Triton X-100) in a total volume of 0.25 ml. The incubations were carried out for 60 min at room temperature. The beads were harvested, washed three times with TNMT, and resuspended in 25 µl of SDS sample buffer. Bound P1 protamine was analyzed on 13% SDS-polyacrylamide gels and detected by Coomassie Blue staining or Western blotting. In the latter case a horseradish peroxidase-conjugated donkey anti-rabbit secondary antibody was used to visualize immunocomplexes by enhanced chemiluminescence (Pierce). Phosphorylation of GST-wtNt and/or P1 protamine was achieved by incubating the respective proteins with GST-SRPK1 (0.3 µg) or 293T cell extracts overexpressing FLAG-SRPK1 in the presence of 0.5 mM ATP for 3 h at 30 °C prior to immobilization on glutathione-Sepharose beads.

His-tagged p32 was tested for binding to the GST fusion proteins following the same procedure. Incubation of the GST-wtNt/p32 complex immobilized on glutathione-Sepharose beads with phosphorylated P1 protamine (by GST-SRPK1 or 293T cell extracts overexpressing FLAG-SRPK1) was also performed in TNMT buffer as described previously. Bound P1 protamine was analyzed on 13% SDS-polyacrylamide gels and detected by Coomassie Blue staining.

Incubation of GST-wtNt or the GST-wtNt/p32 complex immobilized on glutathione-Sepharose beads with rat testis cytosolic extracts (~400 µg of total protein) or 293T cell extracts (~200 µg of total protein) overexpressing FLAG-SRPK1 was performed in PBST (20 mM phosphate buffer, pH 7.4, 150 mM NaCl, 1% Triton X-100, and 0.5 mM phenylmethylsulfonyl fluoride) in a total volume of 0.25 ml for 60 min at room temperature. Bound SRPK1 and FLAG-SRPK1 were analyzed on 10% SDS-polyacrylamide gels and detected by Western blotting using the anti-SRPK1 polyclonal antibody and an alkaline phosphatase-coupled goat anti-rabbit secondary antibody or the M5 anti-FLAG monoclonal antibody and an alkaline phosphatase-coupled goat antimouse secondary antibody, respectively. In both cases, 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium were used as substrates to visualize the immunocomplexes. In vitro phosphorylation assays of GST-wtNt by bound SRPK1 or FLAG-SRPK1 were performed on beads as described previously.

Immunofluorescence Microscopy—Mouse seminiferous tubule segments were isolated in sequential order after stages VII–VIII using the transillumination-assisted microdissection method (24). Cells from the spermatogenic stages were spread onto polylysine-coated slides and allowed to adhere for 20 min at room temperature. The slides were then fixed with 4% paraformaldehyde in PBS for 20 min at room temperature, washed twice with PBS containing 50 mM ammonium chloride, and permeabilized with PBS containing 0.2% Triton X-100 for 5 min. After washing twice with PBS the slides were incubated for 45 min with the primary antibodies (diluted 1:100 in PBS), washed twice with PBS, and incubated for 30 min with the Cy3-conjugated anti-rabbit secondary antibody (1:500 dilution in PBS). The immunostained samples were counterstained with 4',6-diamidino-2-phenylindole dihydrochloride before being subjected to microscopy. The images were collected on a DMLB Leica microscope with an HBO 100-W lamp.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
SRPK1 Phosphorylates LBR and P1 Protamine in Testes—In a previous report, we have detected high levels of LBR kinase activity in testes (10). This activity was found primarily in the cytosol (Fig. 1A; see also Ref. 10). To demonstrate that the cytosolic and nuclear kinase activities targeting LBR, from rat testes, were associated with the same enzyme, we performed in situ kinase assays in SDS-polyacrylamide gels, to which 0.1 mg/ml GST-wtNt (the wild-type nucleoplasmic NH2-terminal domain of LBR; amino acids 1–205) had been added immediately prior to gel polymerization. Fig. 1B shows that, in both cytosolic and nuclear fractions, a protein with a relative molecular mass of ~97 kDa could modify LBR. This was specific because no labeling was detected when GST-wtNt was omitted from the gel or replaced by GST (data not shown). In a following step, GST-wtNt was incubated with rat testis cytosol and recovered by pull-down with glutathione-Sepharose beads. The beads were then used for kinase reactions in the presence of radiolabeled ATP. Phosphorylation of the GST-wtNt protein, detected by autoradiography, suggested the existence of an LBR-associated kinase in testis cytosol (Fig. 1C, left panel). Furthermore, in a similar experiment the complexes recovered with glutathione-Sepharose beads were analyzed by SDS-PAGE and Western blotting using an anti-SRPK1 polyclonal antibody (20). As shown in Fig. 1C (right panel), the 97-kDa protein that could associate with LBR corresponded to the molecular mass of SRPK1.



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  		FIG. 1.
A, subcellular distribution of LBR kinase activity in rat testes. Cytosolic (cyt) and nuclear (nucl) extracts (15 µg) were assayed for LBR kinase activity toward GST-wtNt. The samples were analyzed by SDS-PAGE and autoradiographed. The radioactive bands corresponding to GST-wtNt were excised and measured by scintillation counting. Kinase activity is expressed as units/g of tissue. One unit is the amount of enzyme required to catalyze the transfer of 0.1 nmol of phosphate to 2 µg of GST-wtNt in 30 min at 30 °C. Under the assay conditions no detectable phosphorylation was observed when GST-{Delta}RS was used as substrate. B, in situ kinase assay of cytosolic and nuclear extracts of rat testes (150 µg each). The samples were electrophoresed on a 10% SDS-polyacrylamide gel containing 0.1 mg/ml GST-wtNt, renatured in situ, incubated with [{gamma}-32P]ATP, and subjected to autoradiography. C, GST or GST-wtNt immobilized on glutathione-Sepharose beads were incubated with rat testis cytosol for 1 h at room temperature. The sediments were incubated with [{gamma}-32P]ATP, analyzed by SDS-PAGE, and autoradiographed (left panel). The full-length GST-wtNt migrates with an apparent molecular mass of 51 kDa. The lower bands represent degradation products (see also Ref. 18). Bound SRPK1 was detected by Western blotting using an anti-SRPK1 polyclonal antibody (right panel). D, phosphorylation of purified human P1 protamine by GST-SRPK1. Left panel, Coomassie Blue staining; right panel, autoradiography.

 
SRPK1 also phosphorylated efficiently protamine 1 (Fig. 1D; see also Ref. 10). Consistent with SRPK1 being the major protamine 1 kinase, Ser8 and Ser10 (7RSQSRSR13) were previously identified as the phosphorylation sites of monophosphorylated and diphosphorylated human P1 protamine (6).

Phosphorylation-mediated Association of P1 Protamine with LBR—Given that RS domains are well known for their bridging properties (12), we set out to investigate the potential interaction of P1 protamine with LBR through their RS regions. To this end we tested the ability of purified P1 protamine to bind GST-wtNt and GST-{Delta}RS (the mutant form lacking the RS motifs; 75RSRSRSRSRS84). The complexes were recovered by pull-down with glutathione-Sepharose beads and analyzed by SDS-PAGE and Coomassie Blue staining. As shown in Fig. 2, we were unable to detect any binding of unphosphorylated P1 protamine to the unphosphorylated NH2-terminal domain of LBR (lane 4). However, phosphorylation of P1 protamine by SRPK1 (either GST-SRPK1 or 293T cell extracts overexpressing FLAG-SRPK1) was able to promote its binding to the NH2-terminal domain of LBR (lane 5), whereas no such interaction was obtained with GST-{Delta}RS (lane 8), suggesting that the binding was confined to the RS domain of LBR. The highly basic character of both the NH2-terminal domain of LBR (pI = 10) and P1 protamine (pI > 11) and the fact that binding could still occur under stringent conditions (i.e. in the presence of 0.5 M salt) are in support of a high affinity and specific interaction. Surprisingly, unphosphorylated P1 protamine was unable to bind to the SRPK1-phosphorylated NH2-terminal domain of LBR (lane 6), leading to the conclusion that the phosphorylation of the two RS domains is not functionally equivalent. A minimal interaction was obtained when both RS regions were modified (lane 7).



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  		FIG. 2.
Phosphorylated P1 protamine binds to the RS domain of LBR. Pull-down assays using purified P1 protamine and GST-wtNt or GST-{Delta}RS. GST was included as a control. Bound P1 protamine was detected by Coomassie Blue staining. Phosphorylation of GST-wtNt was achieved by incubating the recombinant protein with GST-SRPK1 (0.3 µg) in the presence of 0.5 mM ATP for 3 h at 30 °C prior to immobilization on glutathione-Sepharose beads. P1 protamine was phosphorylated by GST-SRPK1 under the same assay conditions prior to incubation with the immobilized recombinant proteins. Phosphorylated GST-wtNt and P1 protamine are denoted by asterisks.

 
p32 Protein Prevents Association of both SRPK1 and Protamine 1 with LBR—A polypeptide with an apparent mass of 32 kDa (p32) was previously shown to associate both in vitro and in vivo with the NH2-terminal domain of LBR (16–18). Using in vitro binding assays with GST-wtNt and His-tagged p32, we confirmed the tight interaction between p32 and the RS dipeptides of LBR, only when the latter were unphosphorylated (Fig. 3A). Binding of His-p32 to GST-wtNt was saturable (Fig. 3B) and could still occur in the presence of 0.5 M salt, revealing a high affinity interaction. In this respect, it is noteworthy that p32 was unable to bind to P1 protamine under our assay conditions (data not shown), suggesting that the RS domain of LBR, although indispensable to p32 interaction, may not be sufficient. In favor of this hypothesis, p32 was reported to interact both with the RS domain and with the RNA-binding domain of ASF/SF2, a member of the SR family of splicing factors (15, 25).



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  		FIG. 3.
Binding of p32 to the unphosphorylated RS domain of LBR. A, pull-down assays using His-tagged p32, GST, GST-wtNt, and GST-{Delta}RS. Bound p32 was detected by Coomassie Blue staining. The open circle denotes the position of His-p32. Phosphorylation of GST-wtNt was achieved by incubating the recombinant protein with GST-SRPK1 in the presence of 0.5 mM ATP for 3 h at 30 °C prior to immobilization on glutathione-Sepharose beads. Phosphorylated GST-wtNt is denoted by an asterisk. B, binding of increasing quantities (0, 2, 4, 6, and 10 µg) of His-tagged p32 to GST-wtNt. The arrow denotes the position of His-p32.

 
Because p32 interacts with the RS domain, it may also control the accessibility of LBR to SRPK1. To test this hypothesis GST-wtNt or GST-wtNt/p32 complexes immobilized on glutathione-Sepharose beads were incubated with 293T cell extracts overexpressing FLAG-SRPK1. The beads were then harvested, washed three times with PBST, and used for in vitro phosphorylation assays in the presence of radiolabeled ATP. Bound proteins were also analyzed by Western blotting, using the M5 anti-FLAG monoclonal antibody. As shown in Fig. 4A, binding of p32 completely inhibited binding of SRPK1 (left panel) and the subsequent phosphorylation of the NH2-terminal domain of LBR (right panel).



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  		FIG. 4.
p32 competes with both SRPK1 and phosphorylated P1 protamine for the RS domain of LBR. A, p32 inhibits binding of SRPK1 and the subsequent phosphorylation of GST-wtNt. GST-wtNt immobilized on glutathione-Sepharose beads was preincubated with 0 (buffer only), 4, 6, and 10 µg of p32 and then incubated with 293T cell extracts overexpressing FLAG-tagged SRPK1. The sediments were harvested, washed three times with PBST, and analyzed by SDS-PAGE. Bound SRPK1 was detected by Western blotting using the M5 anti-FLAG monoclonal antibody (left panel). Phosphorylation assays were performed on the beads in the presence of 50 µM [{gamma}-32P]ATP. The samples were analyzed by SDS-PAGE and autoradiographed (right panel). B, p32 prevents binding of phosphorylated P1 protamine to the NH2-terminal domain of LBR. GST-wtNt immobilized on glutathione-Sepharose beads was preincubated with 0 (buffer only), 4, and 10 µg of p32 and then incubated with phosphorylated P1 protamine. Phosphorylation of protamine 1 was achieved by incubating the purified protein with GST-SRPK1 (0.3 µg) in the presence of 0.5 mM ATP for 3 h at 30 °C prior to incubation with GST-wtNt or the GSt-wtNt/p32 complexes. Bound P1 protamine was detected by Coomassie Blue staining. The open circle denotes the position of His-p32.

 
In a following step, to examine whether p32 could also regulate the binding of phosphorylated protamine 1 to GST-wtNt, we performed pull-down assays using GST-wtNt/p32 complexes immobilized on glutathione-Sepharose beads and phosphorylated P1 protamine. Analysis of bound P1 protamine on SDS-polyacrylamide gels followed by Coomassie Blue staining demonstrated that p32 protein prevented the binding of protamine 1 to LBR (Fig. 4B), suggesting that p32 actually competes with both P1 protamine and SRPK1 for the RS domain of LBR.

The NH2-terminal Domain of LBR Pulls Down a Fraction of P1 Protamine and p32 from Testis Nuclear Extracts—To confirm the observed interactions between purified proteins, GST-wtNt immobilized on glutathione-Sepharose beads was incubated with mouse testis nuclear extracts. SDS-PAGE and immunoblotting analysis of the proteins co-sedimenting with the beads showed that a fraction of protamine 1 was able to associate with GST-wtNt (Fig. 5A). The association of p32 could also be readily documented using the same pull-down approach (Fig. 5B).



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  		FIG. 5.
P1 protamine and p32 are precipitated by GST-wtNt from mouse testis nuclear extracts. Nuclear extracts, prepared as described under "Materials and Methods," were adjusted to 0.5 M NaCl and used in pull-down experiments with GST-wtNt or GST alone (control). The bound proteins were analyzed by SDS-PAGE and Western blotting using anti-P1 protamine (A) or anti-p32 (B) rabbit polyclonal antibodies, respectively.

 
Temporal Perinuclear Localization of Protamine 1 and p32 during Mouse Spermiogenesis—To study in detail the distribution of LBR, protamine 1, and p32 during mouse spermiogenesis, several isolated spermiogenic cells during the elongation process were subjected to immunofluorescence microscopy, and a representative analysis is shown in Fig. 6. At the beginning of the elongation process LBR exhibited a peripheral, nuclear envelope distribution (Fig. 6A, a-LBR). With progression of spermiogenesis, a polarization of the signal to one half of the nuclear periphery was apparent (Fig. 6, B and C, a-LBR). Finally, in elongated spermatids the signal was restricted mainly to the posterior pole of the nucleus (Fig. 6D, a-LBR). A similar redistribution was also observed with LAPs (lamina-associated polypeptides), which are also integral proteins of the inner nuclear membrane, and probably reflects the nuclear envelope remodeling during spermiogenesis (26).



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  		FIG. 6.
Immunolocalization of LBR, p32, and P1 protamine in isolated mouse germ cells. Germ cells from various elongation stages were subjected to immunofluorescence microscopy after incubation with rabbit polyclonal antibodies against LBR, p32, and P1 protamine (a-LBR, a-p32, and a-P1) (red). Nuclear staining was achieved by using 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) dye (blue). A, spermatids at the beginning of the elongation process; B, elongating spermatids; C, elongating spermatids isolated from a later stage of the elongation process; D, elongated spermatids.

 
Protamine 1 exhibited the same peripheral, polarized distribution as LBR upon its appearance in the nucleus of elongating spermatids (Fig. 6B, compare a-LBR and a-P1). With progression of the elongation process nuclear labeling increased (Fig. 6C, a-P1), whereas in elongated spermatids the whole nucleus was labeled (Fig. 6D, a-P1), indicating the termination of P1 protamine deposition on sperm chromatin. The redistribution of p32 protein was impressive. p32 exhibited perinuclear localization, similarly to LBR, during the initial stages of the elongating process (Fig. 6A, a-p32). Concomitantly with the appearance of P1 protamine at the nuclear periphery, the perinuclear distribution of p32 was less apparent, and significant labeling of the central area of the nucleus could be observed (Fig. 6B, a-p32). The labeling became exclusively nuclear at a later stage of the elongation process with the first indications of acrosomal localization (Fig. 6C, a-p32), and finally p32 exhibited a clear acrosomal localization in elongated spermatids (Fig. 6D, a-p32). The acrosomal distribution of p32 has been also previously observed, during late spermiogenesis (27), however without any indication of its potential function in this organelle.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study we demonstrated a direct association of LBR with protamine 1 only when the latter was phosphorylated by SRPK1. SRPK1 is predominantly expressed in testes, whereas it is present at low levels in almost all tissues (10). In situ hybridization experiments (10) and Western blotting analysis (data not shown) revealed that SRPK1 is highly expressed in all spermatogenic cells except spermatozoa. The reason for the high level of expression of SRPK1 during the early stages of spermatogenesis may be due to the multiple functions associated with this kinase. SRPK1 was found to regulate through the phosphorylation of SR splicing factors constitutive and alternative pre-mRNA splicing (13) as well as export and translation of mRNAs (28).2 Moreover, SRPK1 was able to mediate the uptake of polyamines through an as yet unidentified signaling pathway (29). Consistent with the diversity of roles displayed by SRPK1 during spermatogenesis, SPK-1 (the Caenorhabditis elegans homologue of SRPK1) is required for germline development in C. elegans, including proliferation, maintenance, and gametogenesis (30).

Postmeiotically, the beginning of spermiogenesis is characterized by a powerful wave of transcription, which is accompanied by high levels of splicing activity (3). Splicing proceeds through phosphorylation-dephosphorylation cycles. Phosphorylation of SR splicing factors is essential for the spliceosome assembly, whereas their dephosphorylation is critical for the catalytic step after spliceosome formation (31). The ASF/SF2 protein is one of the essential members of the SR family of splicing factors. p32 inhibits ASF/SF2 function as a splicing enhancer by preventing stable ASF/SF2 interaction with RNA and also by inhibiting ASF/SF2 phosphorylation by SR protein kinases (15). The sequestration of p32 at the nuclear periphery, via its association with LBR, would therefore allow splicing to proceed unimpaired in early haploid cells. Furthermore, the masking of the RS domain of LBR would prevent its phosphorylation and consequently would leave LBR ready to act as a "docking site" for the protamine 1 molecules.

As spermiogenesis proceeds, histones are sequentially replaced by transition proteins and protamines, resulting in transcriptional silencing. P1 protamine is presumably phosphorylated in the cytoplasm, shortly after its synthesis, considering the predominant cytoplasmic localization of SRPK1 (Ref. 10 and this study). The appearance of phosphorylated P1 protamine in the nucleus is well coordinated with the migration of p32 to the central area of the nucleus and the unmasking of the RS domain of LBR (for a model see Fig. 7). The nature of the event that triggers the dissociation of p32 from the RS domain of LBR still remains obscure.



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  		FIG. 7.
A provisional model illustrating the interactions between the NH2-terminal nucleoplasmic domain of LBR, p32,and P1 protamine. In early haploid cells, p32 is associated with the RS domain of LBR. As spermiogenesis proceeds, an as yet unidentified signal triggers its dissociation from LBR, thereby unmasking the RS domain of LBR and allowing its subsequent binding to phosphorylated protamine 1. LBR may act as a docking site for the replacement of transition proteins (TP) by P1 protamine in certain chromatin layers that come close to the nuclear periphery. Enzymes trapped in the inner nuclear membrane (INM) may also further modify the P1 protamine molecules, thereby facilitating their deposition on sperm chromatin. The detachment of P1 protamine from the nuclear envelope and its tight binding to DNA is postulated to occur through its dephosphorylation.

 
The association of P1 protamine with the nuclear envelope probably represents an important intermediate step prior to its deposition on sperm chromatin. One possibility is that certain layers of the transition protein-organized chromatin come close to the nuclear membrane, and the replacement takes place. Another possibility, not mutually exclusive, is that the nuclear envelope functions as a "working platform" where additional modifications (i.e. methylation) of protamine 1 take place. Those modifications may not only increase the affinity of protamine 1 for sperm DNA but may also recruit specific molecules, such as HP1 (heterochromatin protein 1), that have been shown to be coupled to chromatin condensation and transcriptional silencing (3, 32).

The detachment of protamine 1 from the nuclear envelope and its binding to DNA are probably achieved through its dephosphorylation (Fig. 7). Consistent with this hypothesis, protamines were found mainly dephosphorylated in mature sperm chromatin (5, 6). As spermiogenesis proceeds, the nuclear envelope is sequestered mainly to the posterior pole (preimplantation fossa) of the nucleus (Ref. 26 and this study). Only this specialized region is associated with chromatin in mature sperm. Fractions of P1 protamine that still remain phosphorylated (6) may be responsible for this type of interaction.

Our data clearly show that an integral nuclear envelope protein plays a crucial role in the well orchestrated exchange of basic nuclear proteins during spermiogenesis. Deciphering the molecular machinery that regulates this process and directs it spatially is important, not only from the standpoint of cell biological interest but also for providing valuable insights of biomedical importance.


   FOOTNOTES
 
* This work was supported in part by grants from the Greek Secretariat of Research and Technology (to T. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Laboratory of Biochemistry, Department of Chemistry, Aristotle University of Thessaloniki, 54 006 Thessaloniki, Greece. Tel.: 30-2310-997726; Fax: 30-2310-997689; E-mail: giannako{at}chem.auth.gr.

1 The abbreviations used are: RS, arginine/serine-rich; LBR, lamin B receptor; GST, glutathione S-transferase; PBS, phosphate-buffered saline. Back

2 J. F. Caceres, personal communication. Back


   ACKNOWLEDGMENTS
 
We thank Göran Akusjärvi for the p32 cDNA clone, Philippe Chevaillier for human protamine 1, G. Mosialos for the M5 anti-FLAG monoclonal antibody, Prim Singh for the anti-LBR antibody, and A. Tsiftsoglou for providing us with a tissue culture facility. We also thank S. D. Georgatos and J. G. Georgatsos for valuable discussions.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Hecht, N. B. (1995) Dev. Genet. 16, 95-103[CrossRef][Medline] [Order article via Infotrieve]
  2. Meistrich, M. L. (1989) in Histones and Other Basic Nuclear Proteins (Hnilica, L., Stein, G. S., and Stein, J. L., eds) pp. 165-182, CRC Press, Boca Raton, FL
  3. Sassone-Corsi, P. (2002) Science 296, 2176-2178[Abstract/Free Full Text]
  4. Marushige, Y., and Marushige, K. (1978) Biochim. Biophys. Acta 518, 440-449[Medline] [Order article via Infotrieve]
  5. Oliva, R., and Dixon, G. H. (1991) Prog. Nucleic Acids Res. Mol. Biol. 40, 25-94[Medline] [Order article via Infotrieve]
  6. Chirat, F., Arkhis, A., Martinage, A., Jaquinot, M., Chevaillier, P., and Sautière, P. (1993) Biochim. Biophys. Acta 1203, 109-114[CrossRef][Medline] [Order article via Infotrieve]
  7. Raukas, E., and Mikelsaar R. H. (1999) Bioessays 21, 440-448[CrossRef][Medline] [Order article via Infotrieve]
  8. Lewis, J. D., Song, Y., de Jong, M. M., Bagha, S. M., and Ausio, J. (2003) Chromosoma 111, 473-482[Medline] [Order article via Infotrieve]
  9. Wu, J. Y., Ribar, T. J., Cummings, D. E., Burton, K. A., McKnight, G. S., and Means, A. R. (2000) Nat. Genet. 25, 448-452[CrossRef][Medline] [Order article via Infotrieve]
  10. Papoutsopoulou, S., Nikolakaki, E., Chalepakis, G., Kruft, V., Chevaillier, P., and Giannakouros, T. (1999) Nucleic Acids Res. 27, 2972-2980[Abstract/Free Full Text]
  11. Biggiogera, M., Muller, S., Courtens, J. L., Fakan, S., and Romani, M. G. (1992) Microsc. Res. Tech. 20, 259-267[CrossRef][Medline] [Order article via Infotrieve]
  12. Valcarcel, J., and Green, M. R. (1996) Trends Biochem. Sci. 21, 296-301[CrossRef][Medline] [Order article via Infotrieve]
  13. Gui, J.-F., Lane, W. S., and Fu, X.-D. (1994) Nature 369, 678-682[CrossRef][Medline] [Order article via Infotrieve]
  14. Krainer, A. R., Mayeda, A., Kozak, D., and Binns, G. (1991) Cell 66, 383-394[CrossRef][Medline] [Order article via Infotrieve]
  15. Petersen-Mahrt, S. K., Estmer, C., Öhrmalm, C., Matthews, D. A., Russel, W. C., and Akusjärvi, G. (1999) EMBO J. 18, 1014-1024[CrossRef][Medline] [Order article via Infotrieve]
  16. Simos, G., and Georgatos, S. D. (1992) EMBO J. 11, 4027-4036[Medline] [Order article via Infotrieve]
  17. Simos, G., and Georgatos, S. D. (1994) FEBS Lett. 346, 225-228[CrossRef][Medline] [Order article via Infotrieve]
  18. Nikolakaki, E., Simos, G., Georgatos, S. D., and Giannakouros, T. (1996) J. Biol. Chem. 271, 8365-8372[Abstract/Free Full Text]
  19. Papoutsopoulou, S., Nikolakaki, E., and Giannakouros, T. (1999) Biochem. Biophys. Res. Commun. 255, 602-607[CrossRef][Medline] [Order article via Infotrieve]
  20. Nikolakaki, E., Kohen, R., Hartmann, A. M., Stamm, S., Georgatsou, E. and Giannakouros, T. (2001) J. Biol. Chem. 276, 40175-40182[Abstract/Free Full Text]
  21. Blobel, G., and Potter, V. R. (1966) Science 154, 1662-1665[Abstract/Free Full Text]
  22. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve]
  23. Kameshita, I., and Fujisawa, H. (1989) Anal. Biochem. 183, 139-143[CrossRef][Medline] [Order article via Infotrieve]
  24. Parvinen, M., and Vanha-Perttula, T. (1972) Anat. Res. 174, 435-449[CrossRef][Medline] [Order article via Infotrieve]
  25. Yu, L., Lowernstein, P. M., Zhang, Z., and Green, M. (1995) J. Virol. 69, 3017-3023[Abstract]
  26. Alsheimer, M., Fecher, E., and Benavente, R. (1998) J. Cell Sci. 111, 2227-2234[Abstract]
  27. Soltys, B. J., Kang, D., and Gupta, R. S. (2000) Histochem. Cell Biol. 114, 245-255[Medline] [Order article via Infotrieve]
  28. Cáceres, J. F., Screaton, G. R., and Krainer, A. R. (1998) Genes Dev. 12, 55-66[Abstract/Free Full Text]
  29. Erez, O., and Kahana, C. (2001) Mol. Cell Biol. 21, 175-184[Abstract/Free Full Text]
  30. Kuroyanagi, H., Kimura, T., Kazuhiro, W., Hisamoto, N., Matsumoto, K., and Hagiwara, M. (2000) Mech. Dev. 99, 51-64[CrossRef][Medline] [Order article via Infotrieve]
  31. Mermoud, J. E., Cohen, P. T. W., and Lamond, A. I. (1994) EMBO J. 13, 5679-5688[Medline] [Order article via Infotrieve]
  32. Cheung, P., Allis, C. D., and Sassone-Corsi, P. (2000) Cell 103, 263-271[CrossRef][Medline] [Order article via Infotrieve]

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