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, 140 (4), 737-50

SRPK2: A Differentially Expressed SR Protein-Specific Kinase Involved in Mediating the Interaction and Localization of pre-mRNA Splicing Factors in Mammalian Cells

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SRPK2: A Differentially Expressed SR Protein-Specific Kinase Involved in Mediating the Interaction and Localization of pre-mRNA Splicing Factors in Mammalian Cells

H Y Wang et al. J Cell Biol.

Abstract

Reversible phosphorylation plays an important role in pre-mRNA splicing in mammalian cells. Two kinases, SR protein-specific kinase (SRPK1) and Clk/Sty, have been shown to phosphorylate the SR family of splicing factors. We report here the cloning and characterization of SRPK2, which is highly related to SRPK1 in sequence, kinase activity, and substrate specificity. Random peptide selection for preferred phosphorylation sites revealed a stringent preference of SRPK2 for SR dipeptides, and the consensus derived may be used to predict potential phosphorylation sites in candidate arginine and serine-rich (RS) domain-containing proteins. Phosphorylation of an SR protein (ASF/SF2) by either SRPK1 or 2 enhanced its interaction with another RS domain-containing protein (U1 70K), and overexpression of either kinase induced specific redistribution of splicing factors in the nucleus. These observations likely reflect the function of the SRPK family of kinases in spliceosome assembly and in mediating the trafficking of splicing factors in mammalian cells. The biochemical and functional similarities between SRPK1 and 2, however, are in contrast to their differences in expression. SRPK1 is highly expressed in pancreas, whereas SRPK2 is highly expressed in brain, although both are coexpressed in other human tissues and in many experimental cell lines. Interestingly, SRPK2 also contains a proline-rich sequence at its NH2 terminus, and a recent study showed that this NH2-terminal sequence has the capacity to interact with a WW domain protein in vitro. Together, our studies suggest that different SRPK family members may be uniquely regulated and targeted, thereby contributing to splicing regulation in different tissues, during development, or in response to signaling.

Figures

Figure 8
Figure 8
Northern blotting analysis of SR protein kinases (indicated in the right of each panel) in multiple human tissues (A and B) and cell lines (C–E).
Figure 2
Figure 2
Biochemical characterization of SRPK2. (A) In vitro translated SRPK1 (lane 1) and SRPK2 (lane 2) were immunoprecipitated with anti-FLAG antibody followed by kinase assay on beads using bacterial GST-ASF/SF2 as substrate (lanes 3 and 4). In vitro translated and precipitated kinases (35S-labeled) and phosphorylated GST-ASF/SF2 (32P-labeled) are indicated on the right. (B) Phosphorylation of purified RS domain–containing proteins (∼0.1 μg each) by SRPK2. (C) Phosphorylation of a panel of ASF/SF2 mutants (∼1 μg each) by SRPK2. These mutants were described by Caceres and Krainer (1993) and used in our previous characterization of SRPK1 (Gui et al., 1994b ) and Clk/Sty (Colwill et al., 1996b ). Residual phosphorylation in the RT and RG mutant proteins likely occurred outside the mutagenized region as discussed previously (Gui et al., 1994b ). (D) SRPK2-mediated phosphorylation restored the mAb104 phosphoepitope on bacterial GST-ASF/SF2.
Figure 4
Figure 4
Composition and comparison of P+1 pockets. The pocket composition for PKA, MAPK, and CK2α and preferred amino acids in the P+1 position of their substrates are from Songyang et al. (1996). The assignment of amino acids that form the P+1 pockets for SRPK1 and 2 is based on the sequence alignment shown in Fig. 1 B, and the selected amino acids for SRPK2 are from Fig. 3 B. The deduced P+1 pocket for Clk/Sty is also based on a similar sequence alignment with kinases whose tertiary structures have been determined (not shown). The subdomain locations of individual amino acids in the P+1 pocket and their positions corresponding to those of PKA are indicated.
Figure 1
Figure 1
Sequence of SRPK2. (A) Nucleotide and deduced amino acid sequences of SRPK2 (GenBank/EMBL/DDBJ accession number U88666). Underlined is a proline-rich sequence and boxed are kinase catalytic domains. (B) Sequence comparison with SRPK1 and PKA. Kinase domains are underlined and marked according to Hanks and Quinn (1991). Identical amino acids between SRPK1 and 2 and conserved amino acids in PKA are in bold. Gaps (indicated by dashes) were introduced to optimize the alignment. Amino acids that form the P+1 pocket are numbered according to the PKA nomenclature. Functional loops are highlighted in black boxes. An asterisk indicates Thr197 in PKA and the corresponding amino acids in SRPK1 and 2, and diamonds label the amino acids that form ion pairs with Thr197 in PKA tertiary structure as previously described (Taylor and Radzio-Andzelm, 1994).
Figure 1
Figure 1
Sequence of SRPK2. (A) Nucleotide and deduced amino acid sequences of SRPK2 (GenBank/EMBL/DDBJ accession number U88666). Underlined is a proline-rich sequence and boxed are kinase catalytic domains. (B) Sequence comparison with SRPK1 and PKA. Kinase domains are underlined and marked according to Hanks and Quinn (1991). Identical amino acids between SRPK1 and 2 and conserved amino acids in PKA are in bold. Gaps (indicated by dashes) were introduced to optimize the alignment. Amino acids that form the P+1 pocket are numbered according to the PKA nomenclature. Functional loops are highlighted in black boxes. An asterisk indicates Thr197 in PKA and the corresponding amino acids in SRPK1 and 2, and diamonds label the amino acids that form ion pairs with Thr197 in PKA tertiary structure as previously described (Taylor and Radzio-Andzelm, 1994).
Figure 3
Figure 3
Substrate specificity of SRPK2 determined by peptide selection. (A) The serine-oriented and arginine-directed peptide library used in this study. M = Met, A = Ala, K = Lys, and X = any amino acid except Ser, Thr, Cys, and Trp. The exclusion of Ser and Thr in the library was to ensure phosphorylation at the single serine residue, and omission of Cys and Trp was to avoid problems with sequencing and oxidation. Positions relative to the serine residue are indicated. (B) Each panel shows the relative abundance of amino acids at each position, which is indicated in the upper right corner.
Figure 5
Figure 5
In vitro protein–protein interaction enhanced by SRPK-mediated phosphorylation. Bacterial GST-ASF/SF2 was phosphorylated by SRPK1 (lane 4) or SRPK2 (lane 6) or mock-phosphorylated (lanes 3 and 5). In vitro translated U1 70K (lane 1, total input in each binding assay) was incubated with beads coupled with GST or treated GST-ASF/SF2. Bound proteins were resolved in a 10% SDS-PAGE followed by autoradiography. (A) Coomassie blue staining of total (lane 1) and bound proteins (lanes 2–6). Note that phosphorylated GST-ASF/SF2 displayed a marked mobility shift. (B) An autoradiograph of the same protein gel. U1 70K was not retained by GST (lane 2), but bound to some extent to mock-phosphorylated GST-ASF/SF2 (lanes 3 and 5) and efficiently to phosphorylated GST-ASF/SF2 by SRPK1 (lane 4) and SRPK2 (lane 6).
Figure 6
Figure 6
Localization of SRPK1 and SRPK2 in transfected HeLa tTA cells. FLAG-tagged SRPK1 (a and b) and SRPK2 (d and e) are localized in both the cytoplasm (represented in a and d) and the nucleus (represented in b and e). Predominant cytoplasmic signal was seen with GFP-SRPK1 (c) and GFP-SRPK2 (f), although some punctate nuclear population was visible (see c, for example). When viewed in different focal planes, signals in both the cytoplasm (g) and the nucleus (h) were evident, and the nuclear population (h) colocalized with the B1C8 antigen (i), a marker for nuclear speckles. A merged image appears yellow (j).
Figure 7
Figure 7
Specific induction of redistribution of endogenous splicing factors by overexpression of SRPK1 and SRPK2. The B1C8 antigen (b, d, and f) and SR proteins (h) stained with mAb104 are concentrated in nuclear speckles of untransfected cells. Overexpression of FLAG-tagged SRPK1 (a) and SRPK2 (e and g) induced the redistribution of both the B1C8 antigen (b and f) and SR proteins (h) from speckles to the nucleoplasm. Overexpression of inactive SRPK1 had little effect on SR protein localization (c and d). Reduced signal in kinase-expressed cells likely reflects some loss of solubilized splicing factors during fixation and permeabilization. In contrast, overexpression of FLAG-tagged SRPK2 (i and k) had no effect on the structural integrity of PML-positive PODs detected by 5E10 (j) or the nuclear envelope stained with anti-lamine antibodies (l).

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