Processive phosphorylation of alternative splicing factor/splicing factor 2

Abstract

SR proteins, named for their multiple arginine͞serine (RS) dipeptide repeats, are critical components of the spliceosome, influencing both constitutive and alternative splicing of pre-mRNA. SR protein function is regulated through phosphorylation of their RS domains by multiple kinases, including a family of evolutionarily conserved SR protein-specific kinases (SRPKs). The SRPK family of kinases is unique in that they are capable of phosphorylating repetitive RS domains with remarkable specificity and efficiency. Here, we carried out kinetic experiments specially developed to investigate how SRPK1 phosphorylates the model human SR protein, ASF͞SF2. By using the start-trap strategy, we monitored the progress curve for ASF͞SF2 phosphorylation in the absence and presence of an inhibitor peptide directed at the active site of SRPK1. ASF͞SF2 modification is not altered when the inhibitor peptide (trap) is added with ATP (start). However, when the trap is added first and allowed to incubate for a specific delay time, the decrease in phosphate content of the enzyme-substrate complex follows a simple exponential decline corresponding to the release rate of SRPK1. These data demonstrate that SRPK1 phosphorylates a specific region within the RS domain of ASF͞SF2 by using a fully processive catalytic mechanism, in which the splicing factor remains ''locked'' onto SRPK1 during RS domain modification.B oth the assembly of the spliceosome and the specificity of splice-site selection in mammalian cells require SR proteins, which contain one or two RNA-recognition motifs (RRMs) in the N terminus and a signature domain enriched with arginine͞ serine dipeptide repeats (RS) in the C terminus (1, 2). The SR proteins are phosphorylated and activated by the SRPK and Clk͞Sty families of protein kinases (3)(4)(5). This posttranslational modification is required for translocation of the SR protein from the cytoplasm to the nucleus (6, 7) and recruitment of the SR proteins from nuclear speckles (also known as splicing-factor compartments) to nascent transcripts for cotranscriptional splicing (8-10). Phosphorylated SR proteins are believed to facilitate 5Ј splice-site recognition through interaction with the RS domain in U1-70 K (a component of the U1 small nuclear ribonucleoprotein) and 3Ј splice-site selection by means of the RS domains present in the U2AF heterodimer (11-13). Finally, reversal of the kinase action by protein phosphatases appears to be essential for spliceosome activation and the return of splicing factors to nuclear speckles (5). ASF͞SF2 is a prototypical SR protein with two RRM domains for RNA binding and a Cterminal RS domain for mediating protein-protein interactions during spliceosome assembly (see Fig. 1C). A total of 20 serine residues lie in the RS domain and are potential sites for phosphorylation. Mutational studies have verified that phosphorylation occurs exclusively in the RS domain (3,7,14).Although the importance of phosphorylation of SR proteins is well documented for their biological functions in splici...

Structural basis for nuclear import of splicing factors by human Transportin 3

“…The SR protein family can be further extended by inclusion of a structurally and functionally diverse group of nuclear proteins that possess RS repeats but lack RRM domains (11). The RS domains are processively phosphorylated on their Ser residues by a set of dedicated kinases (12)(13)(14)(15)(16). Phosphorylation of SR proteins is thought to trigger their import into the nucleus and is subsequently required for spliceosome assembly, whereas their dephosphorylation allows for splicing to proceed (17)(18)(19)(20).…”

Statistics of cellular signal transduction as a race to the nucleus by multiple random walkers in compartment/phosphorylation space

“…On each side of the cytonuclear barrier, there are M ϩ 1 species having different levels of phosphorylation. This network topology can generally be interpreted as representing either processive or distributive mechanisms of phosphorylation (14,15). If the subscript i labels a specific order of phosphorylation, e.g., phosphorylation of residue A, followed by residue B, followed by residue C, .…”

Processive phosphorylation: Mechanism and biological importance

“…Recent reports have shown that ASF/SF2 is phosphorylated by SRPK1 and Clk/Sty kinases in a fully processive manner [71,72]. SRPK1 binding to ASF/SF2 is dependent on the phosphorylation state of ASF/SF2 [73].…”

“…SRPK1 binding to ASF/SF2 is dependent on the phosphorylation state of ASF/SF2 [73]. Serine phosphorylation within the Arg-Ser dipeptide produces a patch of alternating positively and negatively charged amino acids, which tether SRPK1 to ASF/SF2 [72]. The processive mechanism for ASF/SF2 was demonstrated using an elegant "start-trap" strategy [72] ( Figure 3A).…”

“…Serine phosphorylation within the Arg-Ser dipeptide produces a patch of alternating positively and negatively charged amino acids, which tether SRPK1 to ASF/SF2 [72]. The processive mechanism for ASF/SF2 was demonstrated using an elegant "start-trap" strategy [72] ( Figure 3A). In this method, ATP and an SRPK1 inhibitor peptide were added simultaneously to the enzyme-substrate complex.…”