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.
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