Regulation and substrate specificity of the SR protein kinase Clk/Sty

Abstract

SR proteins constitute a family of splicing factors that play key roles in both constitutive and regulated splicing in metazoan organisms. The proteins are extensively phosphorylated, and kinases capable of phosphorylating them have been identified. However, little is known about how these kinases function, for example, whether they target specific SR proteins or whether the kinases themselves are regulated. Here we describe properties of one such kinase, Clk/Sty, the founding member of the Clk/Sty family of dual-specificity kinases. Clk/Sty is autophosphorylated on both Ser/Thr and Thr residues, and using both direct kinase assays and SR protein-dependent splicing assays, we have analyzed the effects of each type of modification. We find not only that the pattern of phosphorylation on a specific SR protein substrate, ASF/SF2, is modulated by autophosphorylation but also that the ability of Clk/Sty to recognize different SR proteins is influenced by the extent and nature of autophosphorylation. Strikingly, phosphorylation of ASF/SF2 is sensitive to changes in Tyr, but not Ser/Thr, autophosphorylation while that of SC35 displays the opposite pattern. In contrast, phosphorylation of a third SR protein, SRp40, is unaffected by autophosphorylation. We also present biochemical data indicating that as expected for a factor directly involved in splicing control (but in contrast to recent reports), Clk/Sty is found in the nucleus of several different cell types.

FIG. 1.

Clk/Sty is autophosphorylated on Ser/Thr and Thr. (A) A total of 1 μg each of GST-Clk/Sty (lane 1) and a kinase-inactive mutant (GST-ClkR/Sty; lane 2) was fractionated by 10% SDS-PAGE and stained with Coomassie blue. The species indicated by the asterisk reflects an N-terminal truncation consisting of GST plus an approximately 10-kDa segment of Clk/Sty, which became phosphorylated in the wild-type but not the mutant sample. (B) A total of 0.5 μg of P-, PTP-, and CIP-Clk/Sty (lanes 1 to 3, respectively), was fractionated by 10% SDS-PAGE and stained with Coomassie blue. (C) A total of 20 to 50 ng of P-Clk/Sty (lane 1), PTP-Clk/Sty (lane 2), and CIP-Clk/Sty (lane 3) was fractionated by 10% SDS-PAGE and transferred to nitrocellulose. The blot was probed with antiphosphotyrosine antibody, and proteins were detected by chemiluminescence.

FIG. 2.

Autophosphorylation of Clk/Sty modulates kinase activity towards ASF/SF2 but not SRp40. (A) Increasing amounts of P-Clk/Sty (1, 5, and 20 ng) and equivalent amounts of PTP- and CIP-Clk/Sty were incubated with 1 μg of MBP under kinase conditions. Reactions were stopped by adding 1/2 volume of 3× SDS sample buffer, and proteins were fractionated by 10% SDS-PAGE and detected by autoradiography. (B and C) Increasing amounts of P-Clk/Sty (5, 20, and 100 ng) or equivalent amounts of PTP- and CIP-Clk/Sty were added to 1 μg of ASF/SF2 (B) or SRp40 (C) purified from E. coli and incubated for 30 min at 37°C. Reactions were processed as described for panel A except that proteins were transferred to nitrocellulose following PAGE. The extent of γ-³²P incorporation was visualized by autoradiography (lanes 1 to 10). Subsequently, blots were incubated with the monoclonal antibody mAb104 (lanes 11 to 20) and reactivity was detected by chemiluminescence.

FIG. 3.

P-Clk/Sty is a mixture of Tyr- and Ser/Thr-autophosphorylated forms. (A) Purification scheme of Tyr (PY-Clk/Sty) and Ser/Thr (PS-Clk/Sty) autophosphorylated forms. P-Clk/Sty was initially purified on a glutathione Sepharose column as described by Prasad et al. (47). It was then dialyzed and fractionated on an antiphosphotyrosine column. The flowthrough consisted of Clk/Sty phosphorylated exclusively on Ser/Thr (PS-Clk/Sty), and Tyr autophosphorylated Clk/Sty (PY-Clk/Sty) was eluted specifically with phenyl phosphate. (B) Clk/Sty is a mixture of differentially phosphorylated forms. PY-Clk/Sty (lane 1) and PS-Clk/Sty (lane 2) were fractionated by 10% SDS-PAGE and transferred to nitrocellulose. Blots were processed as described for Fig. 1. (C and D) Increasing amounts of PY-Clk/Sty (lanes 2 to 4) or PS-Clk/Sty (lanes 5 to 7) were incubated with recombinant ASF/SF2 (C) or SRp40 (D) purified from E. coli. Reactions were processed as described for Fig. 2.

FIG. 4.

Clk/Sty is dephosphorylated on Tyr during splicing in nuclear extracts. A total of 20 and 100 ng of P-Clk/Sty (first lane and third through fifth lanes) or PTP-Clk/Sty (sixth through eighth lanes) was added to nuclear extracts (second through fifth, seventh, and eighth lanes) under splicing conditions. Splicing reactions were allowed to proceed for the indicated times. Proteins were precipitated with 10% trichloroacetic acid and washed with acetone, and proteins were fractionated on 10% SDS-PAGE. Western blots were probed with antiphosphotyrosine antibody, and proteins were detected by chemiluminescence.

FIG. 5.

PY- and PS-Clk/Sty differentially affect splicing activated by distinct SR proteins. Increasing amounts (8, 20, and 50 ng) of PY-Clk/Sty (lanes 2 to 4) and PS-Clk/Sty (lanes 5 to 7) were added to splicing reactions performed with S100 extracts in the presence of phosphatase inhibitors and ASF/SF2 (A) and SRp40 (B). Reaction mixtures were deproteinized after 80 min, and RNAs were precipitated with ethanol. RNAs were fractionated by 5% denaturing PAGE and visualized by autoradiography.

FIG. 6.

Ser/Thr and Tyr autophosphorylation independently influence substrate specificity. Increasing amounts (8, 20, and 50 ng) of P-Clk/Sty (lanes 2 to 4), PTP-Clk/Sty (lanes 5 to 7), and CIP-Clk/Sty (lanes 8 to 10) were added to splicing reactions performed with S100 extracts in the presence of phosphatase inhibitors and ASF/SF2 (A) or SC35 (B). Reaction mixtures were deproteinized after 80 min, and RNAs were precipitated with ethanol. RNAs were fractionated by 5% denaturing PAGE and visualized by autoradiography.

FIG. 7.

Exogenous and endogenous Clk/Sty SR protein kinases are present in the nucleus. For detection of exogenously expressed Clk/Sty, human 293 cells on 10-cm-diameter plates were either mock transfected (lanes 1 and 2) or transfected with HA-Clk/Sty (lanes 3 and 4). Cells were collected at 48 h posttransfection, and nuclear (lanes 1 and 3) and cytoplasmic (lanes 2 and 4) fractions were prepared. Protein samples were boiled in sample buffer and fractionated by 10% SDS-PAGE before being transferred to nitrocellulose. Blots were probed with anti-Clk/Sty antibody, and proteins were detected by chemiluminescence. For detection of endogenous Clk/Sty, Neuro 2A (lanes 5 to 7), NIH 3T3 (lanes 8 to 10), and HeLa (lanes 11 to 13) cells were harvested and subcellular fractionation was performed as described above. Whole-cell lysates (lanes 5, 8, and 11), nuclear fractions (lanes 6, 9, and 12), and cytoplasmic fractions (lanes 7, 10, and 13) were resolved by 10% SDS-PAGE and transferred to nitrocellulose. Blots were probed with anti-Clk/Sty antibody, and proteins were detected by chemiluminescence. The asterisks denote a possible cytoplasmic isoform (see text).