title: 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 betagamma-regulators and exists in two splice variants. The long isoform PhLP(L) and the short isoform PhLP(S) differ by the presence or absence of an 83-amino acid N terminus. In isolated biochemical assay systems, PhLP(L) is the more potent Gbetagamma-inhibitor, whereas the functional role of PhLP(S) is still unclear. We now report that in intact HEK 293 cells, PhLP(S) inhibited Gbetagamma-induced inositol phosphate generation with approximately 20-fold greater potency than PhLP(L). Radiolabeling of transfected HEK 293 cells with [(32)P] revealed that PhLP(L) is constitutively phosphorylated, whereas PhLP(S) is not. Because PhLP(L) 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. PhLP(L) was a good CK2 substrate, whereas PhLP(S) and phosducin were not. Progressive truncation and serine/threonine to alanine mutations of the PhLP(L) N terminus identified a serine/threonine cluster (Ser-18/Thr-19/Ser-20) within a small N-terminal region of PhLP(L) (amino acids 5-28) as the site in which PhLP(L) function was modified in HEK 293 cells. In native tissue, PhLP(L) also seems to be regulated by phosphorylation because phosphorylated and non-phosphorylated forms of PhLP(L) were detected in mouse brain and adrenal gland. Moreover, the alternatively spliced isoform PhLP(S) 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.
authors.affiliation: Institute of Pharmacology and Toxicology, University of Würzburg, Versbacher Strasse 9, 97078 Würzburg, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Humrich
authors.forename: Christina
authors.initials: C
authors.lastname: Bermel
authors.forename: Tobias
authors.initials: T
authors.lastname: Grubel
authors.forename: Ursula
authors.initials: U
authors.lastname: Quitterer
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Lohse
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D047390:Casein Kinase II; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D051379:Mice; D018832:Molecular Chaperones; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009928:Organ Specificity; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D018832:Molecular Chaperones; D009419:Nerve Tissue Proteins; C098789:PDCL protein, human; C498319:Pdcl protein, mouse; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases