UC San Diego
Regulation of SR protein substrates by yeast SR protein kinase, Sky1p
- Author(s): Lukasiewicz, Randall Matthew
- et al.
SR proteins play a role in various processes in gene expression: pre-mRNA splicing, mRNA export and translation. The localization and function of SR proteins depends on their phosphorylation state, which is regulated by SR protein kinases (SRPKs). Although no classical SR proteins have been identified in yeast, an SR-like protein Npl3p plays a role in pre-mRNA processing, mRNA export and translation. Yeast SRPK Sky1p regulates Npl3p localization; phosphorylation by Sky1p signals the import of Npl3p into the nucleus. Methylation of Npl3p is also required for nuclear export of Npl3p-containing mRNPs, suggesting that a molecular switch for Npl3p exists between phosphorylation and methylation. Npl3p is a multi- domain protein. The functionality of the N-terminal APQE domain is unknown. The central domain containing two RNA recognition motifs is responsible for RNA binding. Serine 411 of the C-terminal RGG domain is phosphorylated by Sky1p. We have found a kinase docking groove in Sky1p which is conserved from yeast to humans. We also show that the RGG domain of Npl3p contains multiple docking motifs, each containing two or three arginines separated by glycines. These docking motifs interact positively with the Sky1p docking groove facilitating substrate recruitment to the kinase. Kinetic assays suggest that the docking motifs facilitate Sky1p recognition of Npl3p. Docking interactions are also important for efficient phosphorylation. Recent reports show that the RGG domain of Npl3p is methylated which inhibits Sk1yp-mediated phosphorylation. Inhibition is due to the disruption of the docking interactions between Sky1p and the RGG domain of Npl3p. The docking interaction between Sky1p and Npl3p is similar to the mammalian SRPK1/ASF/SF2 docking interaction reported earlier from our laboratory. We show that Sk1yp binds non-native substrate ASF/SF2 with high affinity and also utilizing a processive mechanism of phosphorylation. To date, no Sky1p substrates are shown to be phosphorylated multiple times. However it is possible that Sky1p may utilize this mechanism in vivo with substrate Gbp2p. Sky1p is a constitutively active kinase whose subcellular localization poses as a form of regulation. The catalytic activity of Sky1p is not known to be regulated. We have observed an anion binding pocket through structural studies, and mutation of the pocket- forming residues affects catalysis. We propose that catalytic activity of Sky1p might be modulated in vivo