PubMed Journals: Biochem J
Source: PMID: 7909431
⇦ ⇨ Biochem J. 1994 Apr 1;299 ( Pt 1):309-15.
Phosphorylation by protein kinase C and
cyclic AMP-dependent protein kinase of synthetic
peptides derived from the linker region
of human P-glycoprotein.
Chambers TC(1), Pohl J, Glass DB, Kuo JF.
(1) Department of Pharmacology, Emory University
School of Medicine, Altanta, GA 30322.
Specific sites in the linker region of human
P-glycoprotein phosphorylated by protein
kinase C (PKC) were identified by means
of a synthetic peptide substrate, PG-2,
corresponding to residues 656-689 from this
region of the molecule. As PG-2 has several
sequences of the type recognized by the
cyclic AMP-dependent protein kinase (PKA),
PG-2 was also tested as a substrate for
PKA. PG-2 was phosphorylated by purified
PKC in a Ca2+/phospholipid-dependent manner,
with a Km of 1.3 microM, and to a maximum
stoichiometry of 2.9 +/- 0.1 mol of phosphate/mol
of peptide. Sequence analysis of tryptic
fragments of PG-2 phosphorylated by PKC
identified Ser-661, Ser-667 and Ser-671
as the three sites of phosphorylation. PG-2
was also found to be phosphorylated by purified
PKA in a cyclic AMP-dependent manner, with
a Km of 21 microM, and to a maximum stoichiometry
of 2.6 +/- 0.2 mol of phosphate/mol of peptide.
Ser-667, Ser-671 and Ser-683 were phosphorylated
by PKA. Truncated peptides of PG-2 were
utilized to confirm that Ser-661 was PKC-specific
and Ser-683 was PKA-specific. Further studies
showed that PG-2 acted as a competitive
substrate for the P-glycoprotein kinase
present in membranes from multidrug-resistant
human KB cells. The membrane kinase phosphorylated
PG-2 mainly on Ser-661, Ser-667 and Ser-671.
These results show that human P-glycoprotein
can be phosphorylated by at least two protein
kinases, stimulated by different second-messenger
systems, which exhibit both overlapping
and unique specificities for phosphorylation
of multiple sites in the linker region of
PMCID: PMC1138056 PMID: 7909431 [Indexed