Regulation of Phosducin-like Protein by Casein Kinase 2 and N-terminal Splicing

Jan Humrich,Christina Bermel,Tobias Grübel,Ursula Quitterer,Martin J Lohse

doi:10.1074/jbc.m206347200

Jan Humrich, Christina Bermel + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m206347200

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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.

Highlights

Phosducin-like protein (PhLP)1 was initially cloned as a product of an ethanol-responsive gene in NG108 –15 neuroblastoma x-glioma cells [1]
Inhibition of Inositol Phosphate Signaling in Intact HEK 293 Cells—To investigate the functional role of PhLPL and PhLPS as G␤␥-regulators in living cells, we subcloned the appropriate cDNAs into the pcDNA3 expression vector and determined the inositol phosphate formation stimulated by the transient cotransfection of phospholipase C␤2 (PLC␤2) and G-protein subunits G␤1 and G␥2 as described previously [19, 20]
The short splice variant PhLPS turned out to be more effective than the long splice variant PhLPL, PhLPS interacts with purified G␤␥ less efficiently than PhLPL [5]

Summary

The abbreviations used are

PhLP, phosducin-like protein; PhLPL, long form of PhLP; PhLPS, short form of PhLP; G␤␥, G-protein ␤␥subunit; PKA, cAMP-dependent protein kinase; GRK2, G-protein-coupled receptor kinase 2; HEK, human embryonic kidney; BAPTA-AM, 1,2-bis(O-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid tetra(acetoxymethy)ester; DRB, 5,6-dichloro-1-␤-D-ribofuranylbenzimidazole; CK2, casein kinase 2; CT, C terminus; ANOVA, analysis of variance; ␭-PPase, ␭-protein phosphatase; PLC, phospholipase. It was reported that PhLPS could be purified from cultured bovine chromaffin cells where it might inhibit nicotine-stimulated exocytosis of catecholamines by a pathway involving a G␤␥1⁄7ADP-ribosylation factor 6 complex [6]. These findings suggested that alternative splicing of PhLP might occur in at least one organ or under specific conditions and might play a role in differential functions of the PhLP isoforms. We report that the G␤␥-regulatory function of PhLPL is inhibited in cells by N-terminal phosphorylation via casein kinase 2 (CK2), whereas PhLPS lacks such a regulatory mechanism. Alternative splicing leads to PhLPS, which is not a substrate for CK2 and escapes this inhibitory mechanism

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