Mapping the binding region(s) on Cp for SRPK2.

(A) Arginine-rich domain (ARD) serves as a binding motif for SRPK2. In vitro GST pull-down assay was performed using SRPK2WT, GST-CpY132A, and GST-Cp149Y132A, of which the ARD was truncated. Reactions were analyzed by SDS-PAGE. Free GST was used as a control. Absence of ARD in Cp abolished the binding of SRPK2 (B) Schematic diagram shows the sequences of the ARD in CpY132A. Mutational constructs generated for the study of protein-protein interaction are shown. (C) Arginine-rich motifs in ARD of Cp serve as binding motifs for SRPK2. GST-tagged mutational Cp constructs and His-SRPK2WT were used in the in vitro GST pull-down assay, free GST as a control. Results were analyzed by SDS-PAGE. Mutations of arginine-rich motif 1 in ARD of Cp greatly weakened the binding of SRPK2. (D) Mutations of arginine-rich motifs in ARD of Cp do not have distinct effects on SRPK2 phosphorylation. In vitro kinase assay using [γ-32P] ATP, SRPK2WT and mutational Cp constructs was performed. Samples were analyzed by SDS-PAGE, followed by autoradiography. The phosphorylation content of GST-tagged CpY132A M1-M4 were similar.

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