Cloning and Characterization of an Alternatively Spliced Form of SR Protein Kinase 1 That Interacts Specifically with Scaffold Attachment Factor-B

Abstract

Serine/arginine protein kinases have been conserved throughout evolution and are thought to play important roles in the regulation of mRNA processing, nuclear import, germline development, polyamine transport, and ion homeostasis. Human SRPK1, which was first identified as a kinase specific for the SR family of splicing factors, is located on chromosome 6p21.2-p21.3. We report here the cloning and characterization of SRPK1a, which is encoded by an alternatively processed transcript derived from the SRPK1 gene. SRPK1a contains an insertion of 171 amino acids at its NH 2 -terminal domain and is similar to SRPK1 in substrate specificity and subcellular localization. . Mammalian SRPK1 and SRPK2, which are highly related in sequence, kinase activity, and substrate specificity, were initially purified and cloned on the basis of their ability to phosphorylate members of the SR family of splicing factors in vitro and mediate splicing factor redistribution during the cell cycle (1-4). SR proteins themselves constitute a highly conserved protein family that is intimately involved in the regulation of pre-mRNA splicing and other steps of RNA metabolism (for reviews, see Refs. 5-7). Biochemical studies demonstrated that SR proteins are required at multiple steps in the assembly of the spliceosome, the dynamic RNA-protein complex that catalyzes intron removal (8 -10). Because RS domains are known to participate in protein-protein and protein-RNA interactions during spliceosome assembly, phosphorylation of these domains can modulate interactions involving SR proteins and is, therefore, essential for their function in constitutive splicing (3,11,12). Furthermore, phosphorylation of SR proteins leads to their release from nuclear speckles, in which they are concentrated to active sites of transcription in the nucleoplasm (1, 3, 13-15). Because changes in the intranuclear SR protein concentration play a critical role in determining which of the competing splice sites are selected, phosphorylation can also indirectly control alternative splice site selection (16 -20). Finally, it has been proposed that the formation of complexes between SF2/ASF and SRPKs may modulate the subcellular distribution of SF2/ASF (21).Yet, the lack both of authentic SR proteins in the yeast genome and of alternative mRNA splicing in yeast suggests that these kinases play roles in the regulation of cellular processes in addition to that of mRNA splicing. Indeed, genetic analyses have implicated Dsk1, which is the fission yeast homologue of SRPK1 in the regulation of chromosome segregation at the metaphase/anaphase transition (22). Furthermore, one of the endogenous substrates of Sky1p, in S. cerevisiae, is the RNA binding protein Npl3p, which has been implicated in mRNA transport (23). Sky1p was found to regulate nuclear import of Npl3p by promoting the interaction between Npl3p

Monochloramine induces reorganization of nuclear speckles and phosphorylation of SRp30 in human colonic epithelial cells: role of protein kinase C

“…Currently, little is known about how SR protein kinases are regulated in cells. It has been hypothesized that the activity of these kinases is modulated through mechanisms involving regulation of their subcellular localization (20,33,42). Recently, Mylonis and Giannakouros have found that protein kinase CK2 directly phosphorylates SRPK1 and enhances its activity (32a), suggesting that SR protein kinases are regulated at multiple levels in vivo.…”

The increasing diversity of functions attributed to the SAFB family of RNA-/DNA-binding proteins

“…SREK1 (also known as SRrp86), for example, has been shown to be antagonised by SAFB [88]. A more generalised effect on splicing could be mediated by an inhibitory interaction between SAFB and the SR protein kinase SRPK1, which phosphorylates specific SR proteins [89,90].…”

“…Conversely, weaker interactions may be maintained in the presence of low salt, but there may be a risk of artefactual interactions. [10], hnRNPC [10], hnRNPD [120], hnRNPG [25,84,121], hnRNPI [10], hnRNPK [10,122], hnRNPU [10] SR proteins SRSF1 [33], SRSF7 [26], SRSF9 [26,33], SREK1 [88], SRRM1 [123] SR protein kinase SRPK1 [89] Chromatin CHD1 [72], NCOR [37,71], HDAC3, [37,71] BRG1 [73], Matrin3 [54] Transcription RNAPolII [33], TAF II 68 [124], steroid receptors [67][68][69], P53 [70] Miscellaneous PIAS1 [37], ZO-2 [125], Zbed4 [126] Also, co-immunoprecipitation does not distinguish between direct protein interactions and interactions mediated by 'bridging' molecules, such as other proteins or nucleic acids. Formation of homo-and hetero-dimers of SAFB1 and SAFB2, as well as interactions with several hnRNP proteins, has been observed (see Table 1 for a summary of some of the proteins shown to interact with SAFB proteins).…”

Alternative RNA splicing complexes containing the scaffold attachment factor SAFB2

“…regulators including Tra2␤, SF2/ASF, 9G8, SRp30c, YT521B, SRp86, SLM1, T-STAR (also known as SLM2), hnRNP A1 and hnRNP D (Denegri et al, 2001;Hartmann et al, 1999;Li et al, 2003;Nayler et al, 1998b;Stoss et al, 2001;Weighardt et al, 1999) and with protein kinases which phosphorylate splicing regulators (SRPK1 and CLK2) (Nikolakaki et al, 2001). Besides these yeast two hybrid interactions based on SAFB1 protein, very little is known about the existence of real endogenous protein interactions or heterocomplexes.…”