Regulation of phosducin-like protein by casein kinase 2 and N-terminal splicing

Abstract

Phosducin-like protein (PhLP) is a member of the phosducin family of G-protein βγ-regulators and exists in two splice variants. The long isoform PhLPL and the short isoform PhLPS differ by the presence or absence of an 83-amino acid N terminus. In isolated biochemical assay systems, PhLPL is the more potent Gβγ-inhibitor, whereas the functional role of PhLPS is still unclear. We now report that in intact HEK 293 cells, PhLPS inhibited Gβγ-induced inositol phosphate generation with ∼20-fold greater potency than PhLPL. Radiolabeling of transfected HEK 293 cells with [32p] revealed that PhLPL is constitutively phosphorylated, whereas PHLPS is not. Because PhLPL has several consensus sites for the constitutively active kinase casein kinase 2 (CK2) in its N terminus, we tested the phosphorylation of the recombinant proteins by either HEK cell cytosol in the presence or absence of kinase inhibitors or by purified CK2. PhLPL was a good CK2 substrate, whereas PhLPS and phosducin were not. Progressive truncation and serine/threonine to alanine mutations of the PhLPL N terminus identified a serine/threonine cluster (Ser-18/Thr-19/Ser-20) within a small N-terminal region of PhLPL (amino acids 5-28) as the site in which PhLPL function was modified in HEK 293 cells. In native tissue, PhLPL also seems to be regulated by phosphorylation because phosphorylated and non-phosphorylated forms of PhLPL were detected in mouse brain and adrenal gland. Moreover, the alternatively spliced isoform PhLPS was also found in adrenal tissue. Therefore, the physiological control of G-protein regulation by PhLP seems to involve phosphorylation by CK2 and alternative splicing of the regulator.