Structurally unique yeast and mammalian serine-arginine protein kinases catalyze evolutionarily conserved phosphorylation reactions.

The mammalian serine-arginine (SR) protein, ASF/SF2, contains multiple contiguous RS dipeptides at the C terminus, and approximately 12 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.