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We have recently reported that the Arg
Volume 270,
Number 26,
Issue of June 30, pp. 15844-15852, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Gln
insulin receptor mutation (QK single mutant) alters a conserved motif
(RK motif) immediately next to the key tyrosine phosphorylation sites
(Tyr
, Tyr
, Tyr
) of the
receptor and constitutively activates its kinase and metabolic
signaling. To investigate further the function of the RK motif, we have
expressed two additional mutant insulin receptors: a single mutant, in
which the second basic residue in the RK motif (Lys
) was
substituted (RA mutant); and a double mutant, in which both the Arg and
the Lys residues were replaced with noncharged amino acids (QA mutant).
As compared with the transfected wild-type receptors (WT), both the
single and the double mutant receptors were normally synthetized and
transported to the plasma membrane and bound insulin normally. Whereas
the double mutant receptor exhibited preserved insulin-dependent
autophosphorylation, kinase activity, and 2-deoxyglucose uptake, all of
these functions were grossly impaired in the two single mutant
receptors. Two-dimensional analysis of tryptic phosphopeptides from
receptor
-subunits revealed that decreased autophosphorylation of
the single mutant receptors mainly involved regulatory
Tyr and carboxyl-terminal Tyr
.
At variance with the insulin-stimulated, insulin-independent tyrosine
kinase activity toward poly(Glu-Tyr) 4:1 was increased 3-fold in both
the double and the single mutants. All mutant receptors induced a
2-fold increase in basal 2-deoxyglucose uptake in NIH-3T3 cells.
Treatment of WT transfected cells with
12-O-tetradecanoylphorbol-13-acetate or 8-bromo-cAMP increased
insulin receptor phosphorylation by 3-fold. No phosphorylation was
observed in cells expressing the two single or the double mutant
receptor. Consistently, purified preparations of PKC and PKA
phosphorylated the WT but not the mutant receptors in vitro. A
17-amino acid synthetic peptide encoding the receptor sequence
surrounding the RK motif inhibited phosphorylation of WT insulin
receptors by both protein kinases A and C. A mutant peptide in which
the RK sequence was replaced by QK (to mimic the mutation in the QK
receptor) exhibited no inhibitory effect. Thus, the RK insulin receptor
motif is required for insulin receptor phosphorylation by protein
kinases C and A and may modulate insulin-independent receptor activity.
The RK motif may also have an important structural role in allowing
normal insulin regulation of the kinase.
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