The Studies On The PSR Protein Of Physarum Polycephalum

SR proteins are a group of structurally related, evolutionary conserved non-snRNP splicing factors, which participate in the maturation of the splieosome. These proteins contain one or two RNA recognition motifs and a C-terminal domain rich in Arg-Ser repeats （RS domain）. SR proteins are phosphorylated at numerous serines in the RS domain by the SR-specific protein kinase （SRPK） family of protein kinases. RS domain phosphorylation is necessary for entry of SR proteins into the nucleus, and may also play important roles in alternative splicing, mRNA export, and other processing events. At present, studies regarding SR protein and SRPKs are mainly on higher eukaryotes. In our previous work, we isolated an SR protein kinase PSRPK from Physarum polycephalum, which can phosphorylate the RS domain of ASF/SF2 both in vivo and in vitro. There is no report of SR protein in P. polycephalum.The aim of this study was to clarify whether SR protein exists in lower eukaryotes, such as P. polycephalum, and to explore its phosphorylation mechanism and interacting proteins. Here, a novel cDNA encoding a serine/arginine （SR）-rich protein, designated PSR, was isolated from the true slime mold P. polycephalum by yeast two hybridization and 5’-RLM-RACE. The deduced amino acid （aa） sequence reveals that PSR contains RS repeats at its C-terminus, similar to the conventional PSRPK substrate ASF/SF2. To study the novel protein, we generated a variety of mutant constructs by PCR and site-directed mutagenesis. Autoradiography indicated that the purified recombinant PSR was phosphorylated by PSRPK in vitro, and the SR-rich domain （aa 460–469） in the PSR protein was required for phosphorylation. In addition, removal of the docking motif （aa 424–450） from PSR significantly reduced the overall catalytic efficiency of the phosphorylation reaction. We also found that the conserved ATP-binding region （ABR） ⁶²⁰LGWGHFSTVWLAIDEKNGGREVALK86 and the serine/threonine protein kinases active-site signature （AS） 184IIHTDLKPENVLL¹⁹⁶ of PSRPK played a crucial role in substrate phosphorylation, and Lys⁸⁶ and Asp¹⁸⁸ were crucial for PSRPK phosphorylation of PSR.14-3-3 proteins associate with phosphorylated SRp38 resulting in protection from dephosphorylation under non-stress conditions, but dissociate in response to heat shock （Shi and Manley, 2007）. However, little is known about how the distribution of SR proteins is affected by 14-3-3. To study the interaction between PSR and P14-3-3, we generated a variety of truncated and mutant peptides of PSR and P14-3-3 and co-expression of them in the mammalian cell strain L929 and yeast strain AH109. The distribution of PSR was affected by co-expression with P14-3-3 in the mammalian cell strain L929 using confocal microscopy. Our results demonstrated that P14-3-3 can modulate the distribution of PSR and that the integrated dock motif combined with the RS domain is necessary for PSR interactions with P14-3-3. Furthermore, the peptides 76KGNENHVKRIKEYRNKVEKELSDICQDILNVLD108 （includingα-helix D） and 217YKDSTLIMQLLRDNLTLWTSD237 （includingα-helix I and the phosphorylation site 235ThrSer236） are involved in P14-3-3 interactions with PSR. The phosphorylation site 235ThrSer236 plays a key role in regulating P14-3-3 interactions with phosphorylated PSR, and thereby modulates the distribution of PSR. This study provides further insight into the mechanism of transcriptional regulation of PSR in the true slime mold.