History of the discovery of the serine-arginine protein kinase (SPRK) familyThe first serine-arginine (SR) protein kinase to be purified and characterized was named SRPK1, for SR-protein-specific kinase 1 [1,2]. It was isolated during a search for the activity that phosphorylates SR splicing factors (also named SR proteins) during mitosis. SRPK1 was shown to phosphorylate SR proteins in a cell-cycle regulated manner, to affect SR protein localization and to inhibit splicing when added in large quantities to a cell-free splicing assay [1,2]. The SRPK1 cDNA was cloned, revealing that the Schizosaccharomyces pombe SRPK1 orthologue, Dsk1, had already been cloned and partially characterized as a kinase with cell cycle-dependent phosphorylation and subcellular localization [3]. The SRPK1 and Dsk1 nucleotide sequencing identified a domain interrupting the kinase catalytic site into two structural entities, Serine-arginine protein kinases (SPRKs) constitute a relatively novel subfamily of serine-threonine kinases that specifically phosphorylate serine residues residing in serine-arginine ⁄ arginine-serine dipeptide motifs. Fifteen years of research subsequent to the purification and cloning of human SRPK1 as a SR splicing factor-phosphorylating protein have lead to the accumulation of information on the function and regulation of the different members of this family, as well as on the genomic organization of SRPK genes in several organisms. Originally considered to be devoted to constitutive and alternative mRNA splicing, SRPKs are now known to expand their influence to additional steps of mRNA maturation, as well as to other cellular activities, such as chromatin reorganization in somatic and sperm cells, cell cycle and p53 regulation, and metabolic signalling. Similarly, SRPKs were considered to be constitutively active kinases, although several modes of regulation of their function have been demonstrated, implying an elaborate cellular control of their activity. Finally, SRPK gene sequence information from bioinformatics data reveals that SRPK gene homologs exist either in single or multiple copies in every single eukaryotic organism tested, emphasizing the importance of SRPK protein function for cellular life.Abbreviations CDK, cyclin dependent kinase; Clk, CDK-like kinase; CK2, casein kinase 2; FOXO1, forkhead box protein O1; HBV, hepatitis B virus; HP1, heterochromatin protein 1; Hsp, heat shock protein; LBR, lamin B receptor; NRF-1, nuclear respiratory factor-1; PGC-1, peroxisome proliferator activated receptor c coactivator-1; RS, arginine-serine; SAFB, scaffold attachment factor B; SR, serine-arginine; SRPK, serine-arginine protein kinase.

We have recently shown that heterochromatin protein 1 (HP1) interacts with the nuclear envelope in an acetylationdependent manner. Using purified components and in vitro assays, we now demonstrate that HP1 forms a quaternary complex with the inner nuclear membrane protein LBR and a sub-set of core histones. This complex involves histone H3/H4 oligomers, which mediate binding of LBR to HP1 and crosslink these two proteins that do not interact directly with each other. Consistent with previous observations, HP1 and LBR binding to core histones is strongly inhibited when H3/H4 are modified by recombinant CREB-binding protein, revealing a new mechanism for anchoring domains of under-acetylated chromatin to the inner nuclear membrane.

Previous studies have identified a subassembly of nuclear envelope proteins, termed "the LBR complex." This complex includes the lamin B receptor protein (LBR or p58), a kinase which phosphorylates LBR in a constitutive fashion (LBR kinase), the nuclear lamins A and B, an 18-kDa polypeptide (p18), and a 34-kDa protein (p34/p32). The latter polypeptide has been shown to interact with the HIV-1 proteins Rev and Tat and with the splicing factor 2 (SF2). Using recombinant proteins produced in bacteria and synthetic peptides representing different regions of LBR, we now show that the LBR kinase modifies specifically arginine-serine (RS) dipeptide motifs located at the nucleoplasmic, NH2-terminal domain of LBR and in members of the SR family of splicing factors. Furthermore, we show that the NH2-terminal domain of LBR binds to p34/p32, whereas a mutated domain lacking the RS region does not. Phosphorylation of LBR by the RS kinase completely abolishes binding of p34/p32, suggesting that this enzyme regulates interactions among the components of the LBR complex.

Arginine/serine protein kinases constitute a novel class of enzymes that can modify arginine/serine (RS) dipeptide motifs. SR splicing factors that are essential for pre-mRNA splicing are among the best characterized proteins that contain RS domains. TwoSRprotein-specifickinases, SRPK1 and SRPK2, have been considered as highly specific for the phosphorylation of these proteins, thereby contributing to splicing regulation. However, despite the fact that SR proteins are more or less conserved among metazoa and have a rather ubiquitous tissue distribution we now demonstrate that SRPK1 is predominantly expressed in testis. In situ expression analysis on transverse sections of adult mouse testis shows that SRPK1 mRNA is abundant in all germinal cells but not in mature spermatozoa. RS kinase activity was found primarily in the cytosol and only minimal activity was detected in the nucleus. In a search for testis-specific substrates of SRPK1 we found that the enzyme phosphorylates human protamine 1 as well as a cytoplasmic pool of SR proteins present in the testis. Protamine 1 belongs to a family of small basic arginine-rich proteins that replace histones during the development of mature spermatozoa. The result of this progressive replacement is the formation of a highly compact chromatin structure devoid of any transcriptional activity. These findings indicate that SRPK1 may have a role not only in pre-mRNA splicing, but also in the condensation of sperm chromatin.

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The lamin B receptor (LBR) is an integral protein of the inner nuclear membrane that is modified at interphase by a nuclear envelope-bound protein kinase. This enzyme (RS kinase) specifically phosphorylates arginine-serine dipeptide motifs located at the NH 2 The nuclear lamina is a filamentous meshwork underlying the inner nuclear membrane (1, 2). In most cells this structure is a heteropolymer of type A and B lamins (3) linked to the inner nuclear membrane through integral membrane proteins. These lamin-binding proteins include the lamin B receptor (LBR 1 or p58; Ref. 4) and the lamina-associated polypeptides (5).LBR possesses a long, hydrophilic NH 2 -terminal domain protruding into the nucleoplasm, eight hydrophobic segments that are predicted to span the membrane, and a hydrophilic COOHterminal domain (6, 7). The NH 2 -terminal domain of LBR contains distinct sites for protein kinase A and p34 cdc2 kinase phosphorylation (8, 9) as well as a stretch rich in arginineserine (RS) motifs (10). The RS motifs are specifically modified by a protein kinase that co-isolates with LBR and is part of a multimeric complex (8, 10). This LBR complex also includes the nuclear lamins and three polypeptides with molecular masses of 18 (p18), 150 (p150), and 34 (p34/p32) kDa, respectively (for pertinent information see Refs. 8, 10, and 12). The latter protein has been shown to interact with the splicing factor 2 (SF2) as well as with the HIV-1 proteins Rev and Tat (13-15). Phosphorylation of LBR by the RS kinase completely abolishes binding of p34/p32, suggesting that this enzyme regulates interactions among the components of the LBR complex (11).At the onset of mitosis, the structure of the nuclear envelope is dramatically altered. The nuclear lamina depolymerizes as a result of hyperphosphorylation of the nuclear lamins at specific sites involved in lamin-lamin (16), lamin-chromatin (17), and lamin-membrane (5) interactions. Following depolymerization, the bulk of type A lamins disperse in the cytoplasm, whereas type B lamins remain bound to remnants of the nuclear envelope. At the same time, the nuclear envelope membranes break down into vesicular structures (1). Apart from lamin hyperphosphorylation, Courvalin et al. (9) also reported that LBR is phosphorylated on serine and threonine residues during mitosis.As the events responsible for nuclear membrane breakdown are not completely understood and in light of the fact that LBR is phosphorylated by the RS kinase during interphase, we found it important to examine the specific modifications of LBR during mitosis. Results presented below reveal that during mitosis LBR is phosphorylated by both RS and p34 cdc2 protein kinases.

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 NH 2 -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.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 NH 2 -terminal domain. In the present study we demonstrate a direct a...

Serine/arginine protein kinases have been conserved throughout evolution and are thought to play important roles in the regulation of mRNA processing, nuclear import, germline development, polyamine transport, and ion homeostasis. Human SRPK1, which was first identified as a kinase specific for the SR family of splicing factors, is located on chromosome 6p21.2-p21.3. We report here the cloning and characterization of SRPK1a, which is encoded by an alternatively processed transcript derived from the SRPK1 gene. SRPK1a contains an insertion of 171 amino acids at its NH 2 -terminal domain and is similar to SRPK1 in substrate specificity and subcellular localization. . Mammalian SRPK1 and SRPK2, which are highly related in sequence, kinase activity, and substrate specificity, were initially purified and cloned on the basis of their ability to phosphorylate members of the SR family of splicing factors in vitro and mediate splicing factor redistribution during the cell cycle (1-4). SR proteins themselves constitute a highly conserved protein family that is intimately involved in the regulation of pre-mRNA splicing and other steps of RNA metabolism (for reviews, see Refs. 5-7). Biochemical studies demonstrated that SR proteins are required at multiple steps in the assembly of the spliceosome, the dynamic RNA-protein complex that catalyzes intron removal (8 -10). Because RS domains are known to participate in protein-protein and protein-RNA interactions during spliceosome assembly, phosphorylation of these domains can modulate interactions involving SR proteins and is, therefore, essential for their function in constitutive splicing (3,11,12). Furthermore, phosphorylation of SR proteins leads to their release from nuclear speckles, in which they are concentrated to active sites of transcription in the nucleoplasm (1, 3, 13-15). Because changes in the intranuclear SR protein concentration play a critical role in determining which of the competing splice sites are selected, phosphorylation can also indirectly control alternative splice site selection (16 -20). Finally, it has been proposed that the formation of complexes between SF2/ASF and SRPKs may modulate the subcellular distribution of SF2/ASF (21).Yet, the lack both of authentic SR proteins in the yeast genome and of alternative mRNA splicing in yeast suggests that these kinases play roles in the regulation of cellular processes in addition to that of mRNA splicing. Indeed, genetic analyses have implicated Dsk1, which is the fission yeast homologue of SRPK1 in the regulation of chromosome segregation at the metaphase/anaphase transition (22). Furthermore, one of the endogenous substrates of Sky1p, in S. cerevisiae, is the RNA binding protein Npl3p, which has been implicated in mRNA transport (23). Sky1p was found to regulate nuclear import of Npl3p by promoting the interaction between Npl3p