Novel SR-rich-related Protein Clasp Specifically Interacts with Inactivated Clk4 and Induces the Exon EB Inclusion of Clk

Abstract

We identified a novel serine/arginine (SR)-rich-related protein as a binding partner of Clk4 mutant lacking kinase activity (Clk4 K189R) in the two-hybrid screen and designated it Clasp (Clk4-associating SR-related protein). Northern blot analysis revealed that Clasp mRNA was highly expressed in brain, and in situ hybridization of a mouse brain sagittal section hybridized with antisense probes revealed that both Clasp and Clk4 mRNAs were expressed in the hippocampus, the cerebellum, and the olfactory bulb. Two forms of Clasp were produced by a frameshift following alternative splicing. The staining of an HA-tagged short form of Clasp (ClaspS) showed a nucleoplasmic pattern, while the long form of Clasp (ClaspL) was localized as nuclear dots. In vitro protein interaction assay demonstrated that Clk4 K189R was bound to Clasp while wild Clk4 was not. Overexpression of ClaspL promoted accumulation of Clk4 K189R in the nuclear dots and the exon EB inclusion from CR-1 and CR-2 pre-mRNA of Clk1. These data suggest that Clasp is a binding partner of Clk4 and may be involved in the regulation of the activity of Clk kinase family.Serine/arginine (SR)-rich proteins are essential splicing factors (1) that promote splice-site recognition (2) at an early stage of spliceosome assembly and also influence the selection of alternative splice sites (3-5). They exist in phosphorylated forms in cells, and the phosphorylation state of SR proteins appears to influence their activities in general and alternative splicing (6, 7), as well as their subnuclear localization and nuclear-cytoplasmic transport properties (7-9). Nuclear speckles are subnuclear regions where SR proteins and other splicing components are concentrated, and they are thought to represent sites of storage or assembly for splicing factors (10). SR protein-specific kinase (SRPK) 1 1 was originally identified as a kinase of SC35 in extracts from HeLa cells (11,12). Addition of purified SRPK1 to permeabilized cells or overexpression of SRPK1, 2, or CDC2-like kinase (Clk)1 in transfected cells result in an apparent disassembly of the nuclear speckles (11,(13)(14)(15)(16). These results suggest that phosphorylation or hyperphosphorylation causes release of these factors from the speckles or that perhaps that the integrity of these structures is compromised.Clk1 was initially cloned as a CDC2-like kinase (17). Clk1 has an arginine/serine (RS) domain at its N terminus and interacts with several members of the SR protein family and SR-related protein in a yeast two-hybrid screen (13, 18). Clk1 phosphorylates SR proteins and affects SR protein-dependent splicing (6). In mammals, Clk is a member of a protein kinase subfamily that contains at least four isoforms Clk1, Clk2, Clk3, and Clk4 (19,20). mRNAs for all four Clk isoforms are alternatively spliced to produce proteins in which the kinase domain is missing (16, 21). Clk1 was independently isolated with antiphosphotyrosine antibody screens as a prototypical dual-specific kinase, termed STY (22, 23). Clk autophosphorylates w...

Regulation and Substrate Specificity of the SR Protein Kinase Clk/Sty

“…RS domain phosphorylation has significant, protein-specific effects on protein-protein interactions (62,63). Indeed, a novel RS domain containing CLASP, a protein that interacts with unphosphorylated (i.e., kinase-inactive) but not phosphorylated Clk4, was recently described (26). Such interactions could affect kinase activity and autophosphorylation in a number of ways, for example, by facilitating or interfering with recognition of specific residues or by directly influencing the structure and activity of the catalytic domain (25).…”

Manipulation of Alternative Splicing by a Newly Developed Inhibitor of Clks

“…The position of different spliced products is indicated. screening (49). TG003, a specific inhibitor of Clk1/Sty and Clk4, will be a valuable tool to dissect the regulatory mechanisms involving SR protein phosphorylation in vivo and may be applicable for the therapeutic manipulation of abnormal splicing.…”

Pnn and SR family proteins are differentially expressed in mouse central nervous system

“…SF2/ASF, another genuine SR protein, however, was expressed at lower levels in the brain (Hanamura et al 1998). Nevertheless, Clasp (Clk4-associating SR-related protein), like SC35, displayed high expression level in hippocampus, cerebellum, and olfactory bulb (Katsu et al 2002). These investigations hinted that various types of tissues might differentially express certain kinds of SR or SR-related proteins, but focused on distinct regions in the CNS.…”

“…For example, higher levels of expression are found in the brain with lower levels in the liver (Black 2003;Grabowski and Black 2001;Katsu et al 2002). During the mouse embryonic development, the expression levels of pinin message varied in different tissues (Leu and Ouyang 2006).…”