Structurally Unique Yeast and Mammalian Serine-Arginine Protein Kinases Catalyze Evolutionarily Conserved Phosphorylation Reactions

Abstract

The mammalian serine-arginine (SR) protein, ASF/SF2, contains multiple contiguous RS dipeptides at the C terminus, and ϳ12 of these serines are processively phosphorylated by the SR protein kinase 1 (SRPK1). We have recently shown that a docking motif in ASF/SF2 specifically interacts with a groove in SRPK1, and this interaction is necessary for processive phosphorylation. We previously showed that SRPK1 and its yeast ortholog Sky1p maintain their active conformations using diverse structural strategies. Here we tested if the mechanism of ASF/SF2 phosphorylation by SRPK is evolutionarily conserved. We show that Sky1p forms a stable complex with its heterologous mammalian substrate ASF/SF2 and processively phosphorylates the same sites as SRPK1. We further show that Sky1p utilizes the same docking groove to bind yeast SR-like protein Gbp2p and phosphorylates all three serines present in a contiguous RS dipeptide stretch. However, the mechanism of Gbp2p phosphorylation appears to be non-processive. Thus, there are physical attributes of SR and SR-like substrates that dictate the mechanism of phosphorylation, whereas the ability to processively phosphorylate substrates is inherent to SR protein kinases.Pre-mRNA splicing and mRNA export are complex processes, which involve a large number of protein and RNA factors (1-4). SR proteins, a class of non-small nuclear ribonucleoprotein splicing factors, participate in every step in the spliceosome assembly and catalysis (5). SR proteins have unique domain architecture. The N-terminal domain contains one or two RNA recognition motifs (RRMs) 3 and is responsible for RNA binding, and the C-terminal domain, known as the RS domain, is rich in long stretches of serine-arginine/arginineserine (SR/RS) dipeptides (6). The RS domains of SR proteins are extensively phosphorylated. Although many kinases have been demonstrated to target SR proteins, members of the SR protein kinase (SRPK) family have been established as the predominant kinase (6 -10). Mammalian SR protein kinase 1 (SRPK1) and the yeast enzyme, Sky1p, are the two most studied SRPKs. Members of the SRPK family display strict substrate specificity, preferring to phosphorylate only serine residues flanked by arginines. One of the well studied mammalian SR proteins, ASF/SF2, contains 20 serines within its 50-residuelong RS domain (see Fig. 1a). SRPK1 phosphorylates ϳ10 -12 of these serines in the N-terminal RS1 region of the RS domain (11). This multisite phosphorylation occurs in a processive manner where the kinase remains bound to the substrate until all sites are phosphorylated (12). Phosphorylation of RS1 is essential for the nuclear import of ASF/SF2.In yeast, Sky1p regulates the phosphorylation and nuclear import of SR-like proteins . However, unlike the mammalian SR proteins, the yeast proteins do not have a classic RS domain with long stretches of arginine/ serine repeats (6, 16) (Fig. 1a). In the case of Npl3p, eight isolated RS dipeptides are present within the RGG domain, and the C-terminal most RS dipeptide is...

Regulation of SR protein phosphorylation and alternative splicing by modulating kinetic interactions of SRPK1 with molecular chaperones

“…Our previous work showed that SRPK1 is a constitutively active kinase (Nolen et al 2001;Lukasiewicz et al 2007) and that an accessory domain (a spacer sequence that splits conserved kinase domains into two blocks) is involved in partitioning of the kinase between the cytoplasm and nucleus, suggesting that the cellular distribution of the kinase, rather than activity, is subject to regulation (Ding et al 2006). We now show that SRPK1 directly interacts with two specific cochaperones for major heat-shock proteins and that the ATPase activity of Hsp90 plays a critical role in regulating the cellular distribution of the kinase in the cell.…”

Pharmacology of Modulators of Alternative Splicing

“…The domain arrangement in this family of kinases is unique; in fact they contain a large insert, called the spacer domain, that bifurcates the kinase core. The space domain plays a key role in the subcellular localization of these kinases (Lukasiewicz et al, 2007). …”

“…The C terminus is basic, with 24% of the residues being prolines (Gallo et al, 1994). The two ends of the previously mentioned space domain fold into unique helical structures and interact with the kinase core (Lukasiewicz et al, 2007). …”

A novel <italic>Physarum polycephalum</italic> SR protein kinase specifically phosphorylates the RS domain of the human SR protein, ASF/SF2

“…These sites are located within the CD at the N-terminus. Crystal structure analysis of Sky1p and SRPK1 showed that the tertiary structures of the two CDs are conserved from yeast to humans and that they share a similar active center conformation composed of the ATP-binding region and an active site [11][12][13]. SWISS-MODEL software [26][27][28] was used to visualize the tertiary structure of PSRPK (Fig.…”

“…Research on SRPKs has predominantly focused on human SRPK1 and yeast Sky1p. These two SRPKs share a similar crystal structure, and the active center is located in the conserved, N-terminal domain, and both can phosphorylate the RS dipeptide stretch of the SR protein, ASF/SF2, in a processive manner [11][12][13]. SRPKs can also phosphorylate lamin B receptor, protamine 1, hepatitis B virus core protein, and the C-terminal half of the cisplatin resistance-associated overexpressed protein [14][15][16][17].…”