The arginine-serine (RS)-rich domain of the SR protein ASF/SF2 is phosphorylated by SR protein kinases (SRPKs) and Clk/Sty kinases. However, the mode of phosphorylation by these kinases and their coordination in the biological regulation of ASF/SF2 is unknown. Here, we report the crystal structure of an active fragment of human SRPK1 bound to a peptide derived from an SR protein. This structure led us to identify a docking motif in ASF/SF2. We find that this docking motif restricts phosphorylation of ASF/SF2 by SRPK1 to the N-terminal part of the RS domain - a property essential for its assembly into nuclear speckles. We further show that Clk/Sty causes release of ASF/SF2 from speckles by phosphorylating the C-terminal part of its RS domain. These results suggest that the docking motif of ASF/SF2 is a key regulatory element for sequential phosphorylation by SRPK1 and Clk/Sty and, thus, is essential for its subcellular localization.

Assembly of the spliceosome requires the participation of SR proteins, a family of splicing factors rich in arginine-serine dipeptide repeats. The repeat regions (RS domains) are polyphosphorylated by the SRPK and Clk/Sty families of kinases. The two families of kinases have distinct enzymatic properties, raising the question of how they may work to regulate the function of SR proteins in RNA metabolism in mammalian cells. Here we report the first mass spectral analysis of the RS domain of ASF/SF2, a prototypical SR protein. We found that SRPK1 was responsible for efficient phosphorylation of a short stretch of amino acids in the N-terminal portion of the RS domain of ASF/SF2 while Clk/Sty was able to transfer phosphate to all available serine residues in the RS domain, indicating that SR proteins may be phosphorylated by different kinases in a stepwise manner. Both kinases bind with high affinity and use fully processive catalytic mechanisms to achieve either restrictive or complete RS domain phosphorylation. These findings have important implications on the regulation of SR proteins in vivo by the SRPK and Clk/Sty families of kinases.It is estimated that more than one-half of human genes are alternatively spliced (1). Although many of these genes may contain only a few splice variants, a few have been shown to possess thousands (1-3). RNA splicing occurs in the nucleus at a macromolecular complex composed of many RNA and protein molecules known as the spliceosome (4, 5). In the last decade it has become apparent that both the assembly of the spliceosome and selection of splice sites depend on the proper phosphorylation of a family of splicing factors known as SR proteins (1). The SR proteins are so named because they have long stretches of arginineserine dipeptide repeats in RS domains. Phosphorylation leads to translocation of the SR protein from the cytoplasm to the nucleus (6, 7) and recruitment of the SR proteins from sites of nuclear storage in speckles to nascent transcripts for splicing (8 -10). Phosphorylated SR proteins are believed to facilitate both 5Ј and 3Ј splice site recognition through interaction with the RS domain in U1-70 K (a component of the U1 small nuclear ribonucleoprotein) and the U2AF heterodimer (11-13). More recently, RS domains were shown to interact directly with critical cis-acting elements in pre-mRNA (14,15). In addition to their functions in the spliceosome, SR proteins are also shown to play a role in the export of processed mRNA from the nucleus to the cytoplasm for translation. Two shuttling SR proteins, ASF/SF2 6 and 9G8, have been shown to conduct the export of mRNA through interactions with a nuclear export factor, TAP (16 -18). Hypophosphorylation of these SR proteins promotes interaction with TAP suggesting that transport may require splicing factor dephosphorylation. These shuttling SR proteins were also found to play a direct role in translation in the cytoplasm (19).Although it is clear that SR proteins can be polyphosphorylated in the RS domain regions by the SRPK ...

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SR proteins and related RS domain-containing polypeptides are an important class of splicing regulators in higher eukaryotic cells. The RS domain facilitates nuclear import of SR proteins and mediates protein-protein interactions during spliceosome assembly; both functions appear to subject to regulation by phosphorylation. Previous studies have identified two nuclear import receptors for SR proteins, transportin-SR1 and transportin-SR2. Here we show that transportin-SR1 and transportin-SR2 are the alternatively spliced products of the same gene and that transportin-SR2 is the predominant transcript in most cells and tissues examined. While both receptors import typical SR proteins in a phosphorylation-dependent manner, they differentially import the RS domain-containing splicing regulators hTra2␣ and hTra2␤ in different phosphorylation states. We suggest that differential regulation of nuclear import may serve as a mechanism for homeostasis of RS domain-containing splicing factors and regulators in the nucleus and for selective cellular responses to signaling.Small nuclear ribonucleoprotein particles and a large number of protein factors are required for the assembly of spliceosomes, where pre-mRNA is processed into mature mRNA (1, 2). SR proteins are a family of non-small nuclear ribonucleoprotein particles splicing factors that are important for both constitutive and regulated splicing (3). Typical SR proteins contain one or two RNA recognition motifs, which are responsible for their sequence-specific RNA binding activities, and an RS domain enriched with arginine and serine repeats in the C terminus (4). Many biochemically and genetically identified splicing regulators, such as Tra and Tra2 in the Drosophila sex determination pathway, are related to SR proteins by the presence of an RS domain (3, 4). Interestingly, two Tra2 homologues, hTra2␣ and hTra2␤, appear to function as general sequencespecific splicing activators in mammalian cells (5).All SR proteins are extensively modified by phosphorylation, which has been shown to be critical for RS domain-mediated protein-protein interactions important for spliceosome assembly (6 -8). Both hypo-and hyperphosphorylation, however, appear to be inhibitory to the function of SR proteins in splicing (9), but the precise mechanism for why a hyperphosphorylated SR is inhibitory remains to be understood, especially in light of a recent observation that RS repeats in the RS domain of the SR protein ASF/SF2 can be substituted by RE or RD to mimic the hyperphosphorylation state in splicing (10). This may be related to the question whether modulation of SR protein function by phosphorylation is due to changes in overall charge distribution brought by phosphorylation in the RS domain, or more specifically, to the modification at some critical sites, or perhaps, to both.SR proteins are distributed in a speckled pattern in interphase nuclei and become dispersed throughout the cell during mitosis as a result of hyperphosphorylation (11). More recently, hyperphosphorylated SR pr...

The mammalian serine-arginine (SR) protein, ASF/SF2, contains multiple contiguous RS dipeptides at the C terminus, and ϳ12 of these serines are processively phosphorylated by the SR protein kinase 1 (SRPK1). We have recently shown that a docking motif in ASF/SF2 specifically interacts with a groove in SRPK1, and this interaction is necessary for processive phosphorylation. We previously showed that SRPK1 and its yeast ortholog Sky1p maintain their active conformations using diverse structural strategies. Here we tested if the mechanism of ASF/SF2 phosphorylation by SRPK is evolutionarily conserved. We show that Sky1p forms a stable complex with its heterologous mammalian substrate ASF/SF2 and processively phosphorylates the same sites as SRPK1. We further show that Sky1p utilizes the same docking groove to bind yeast SR-like protein Gbp2p and phosphorylates all three serines present in a contiguous RS dipeptide stretch. However, the mechanism of Gbp2p phosphorylation appears to be non-processive. Thus, there are physical attributes of SR and SR-like substrates that dictate the mechanism of phosphorylation, whereas the ability to processively phosphorylate substrates is inherent to SR protein kinases.Pre-mRNA splicing and mRNA export are complex processes, which involve a large number of protein and RNA factors (1-4). SR proteins, a class of non-small nuclear ribonucleoprotein splicing factors, participate in every step in the spliceosome assembly and catalysis (5). SR proteins have unique domain architecture. The N-terminal domain contains one or two RNA recognition motifs (RRMs) 3 and is responsible for RNA binding, and the C-terminal domain, known as the RS domain, is rich in long stretches of serine-arginine/arginineserine (SR/RS) dipeptides (6). The RS domains of SR proteins are extensively phosphorylated. Although many kinases have been demonstrated to target SR proteins, members of the SR protein kinase (SRPK) family have been established as the predominant kinase (6 -10). Mammalian SR protein kinase 1 (SRPK1) and the yeast enzyme, Sky1p, are the two most studied SRPKs. Members of the SRPK family display strict substrate specificity, preferring to phosphorylate only serine residues flanked by arginines. One of the well studied mammalian SR proteins, ASF/SF2, contains 20 serines within its 50-residuelong RS domain (see Fig. 1a). SRPK1 phosphorylates ϳ10 -12 of these serines in the N-terminal RS1 region of the RS domain (11). This multisite phosphorylation occurs in a processive manner where the kinase remains bound to the substrate until all sites are phosphorylated (12). Phosphorylation of RS1 is essential for the nuclear import of ASF/SF2.In yeast, Sky1p regulates the phosphorylation and nuclear import of SR-like proteins . However, unlike the mammalian SR proteins, the yeast proteins do not have a classic RS domain with long stretches of arginine/ serine repeats (6, 16) (Fig. 1a). In the case of Npl3p, eight isolated RS dipeptides are present within the RGG domain, and the C-terminal most RS dipeptide is...