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 ␤␥-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 G␤␥-inhibitor, whereas the functional role of PhLP S is still unclear. We now report that in intact HEK 293 cells, PhLP S inhibited G␤␥-induced inositol phosphate generation with ϳ20-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.

Function of phosducin-like proteins in G protein signaling and chaperone-assisted protein folding

“…When over-expressed PhLP1 s blocked Gβ and Gγ expression and strongly inhibited Gβγ signaling [17,84], as would be predicted by the effects of PhLP1 Δ1-75 on Gβγ assembly. It is noteworthy that very little PhLP1 s is found in most tissues, but significant amounts are found in the adrenal gland [25,84]. This expression pattern for PhLP1 s is strikingly similar to the tissue distribution of PhLP1 S18-20 phosphorylation.…”

“…This expression pattern for PhLP1 s is strikingly similar to the tissue distribution of PhLP1 S18-20 phosphorylation. PhLP1 is completely phosphorylated within the S18-20 site in most tissues, but in the adrenal gland this site is predominantly dephosphorylated [84]. These observations suggest that Gβγ synthesis in the adrenal gland may be down-regulated significantly by these PhLP1 species.…”

“…Interestingly, the PhLP1 Δ1-75 variant is very similar to a naturally occurring PhLP1 truncation (designated PhLP1 s ) that is missing the N-terminal 83 amino acids due to alternative mRNA splicing [24,84]. When over-expressed PhLP1 s blocked Gβ and Gγ expression and strongly inhibited Gβγ signaling [17,84], as would be predicted by the effects of PhLP1 Δ1-75 on Gβγ assembly. It is noteworthy that very little PhLP1 s is found in most tissues, but significant amounts are found in the adrenal gland [25,84].…”

“…Initially, it was reported that PhLP1 was phosphorylated by CK2 within the S18-20 cluster and that an S18-20A alanine substitution variant was more effective at inhibiting Gβγ signaling than wildtype PhLP1 [84]. However, it was unclear why the PhLP1 S18-20A variant was a better inhibitor given that CK2 phosphorylation of PhLP1 did not change its binding affinity for Gβγ [65].…”

“…As a result, PhLP1 Δ1-75 could not be phosphorylated and bound Gβγ poorly, yet it maintained its full CCT binding capacity [18]. Interestingly, the PhLP1 Δ1-75 variant is very similar to a naturally occurring PhLP1 truncation (designated PhLP1 s ) that is missing the N-terminal 83 amino acids due to alternative mRNA splicing [24,84]. When over-expressed PhLP1 s blocked Gβ and Gγ expression and strongly inhibited Gβγ signaling [17,84], as would be predicted by the effects of PhLP1 Δ1-75 on Gβγ assembly.…”