Natural variants of human p85 alpha phosphoinositide 3-kinase in severe insulin resistance: a novel variant with impaired insulin-stimulated lipid kinase activity.
OBJECTIVE
Phosphoinositide 3-kinase (PI 3K) plays a central part in the mediation of insulin-stimulated glucose disposal. No genetic studies of this enzyme in human syndromes of severe insulin resistance have been previously reported.


METHODS
Phosphoinositide 3-kinase p85 alpha regulatory subunit cDNA was examined in 20 subjects with syndromes of severe insulin resistance by single strand conformational polymorphism and restriction fragment length polymorphism analyses. Insulin-stimulated phosphoinositide 3-kinase activity and recruitment into phosphotyrosine complexes of variants of p85 alpha were studied in transiently transfected HEK293 cells. Phosphopeptide binding characteristics of wild-type and mutant p85 alpha-GST fusion proteins were examined by surface plasmon resonance.


RESULTS
The common p85 alpha variant, Met326I1e, was identified in 9 of the 20 subjects. Functional studies of the Met326Ile variant showed it to have equivalent insulin-stimulated lipid kinase activity and phosphotyrosine recruitment as wild-type p85 alpha. A novel heterozygous mutation, Arg409Gln, was detected in one subject. Within the proband's family, carriers of the mutation had a higher median fasting plasma insulin (218 pmol/l) compared with wild-type relatives (72 mol/l) (n = 8 subjects, p = 0.06). The Arg409Gln p85 alpha subunit was associated with lower insulin-stimulated phosphoinositide 3-kinase activity compared with wild-type (mean reduction 15%, p < 0.05, n = 5). The recruitment of Arg409Gln p85 alpha into phosphotyrosine complexes was not significantly impaired. GST fusion proteins of wild-type and mutant p85 alpha showed identical binding to phosphopeptides in surface plasmon resonance studies.


CONCLUSIONS
Mutations in p85 alpha are uncommon in subjects with syndromes of severe insulin resistance. The Met326Ile p85 alpha variant appears to have no functional effect on the insulin-stimulated phosphoinositide 3-kinase activity. The impaired phosphoinositide 3-kinase activity of the Arg409Gln mutant suggests that it could contribute to the insulin resistance seen in this family.
Baynes
K C
KC
Department of Medicine, University of Cambridge, UK.
Beeton
C A
CA
Panayotou
G
G
Stein
R
R
Soos
M
M
Hansen
T
T
Simpson
H
H
O'Rahilly
S
S
Shepherd
P R
PR
Whitehead
J P
JP
D000328:Adult; D001483:Base Sequence / Q000235:genetics; D002460:Cell Line; D005215:Fasting / Q000097:blood; D005260:Female; D014644:Genetic Variation; D006801:Humans; D007328:Insulin / Q000097:blood / Q000494:pharmacology; D007333:Insulin Resistance / Q000235:genetics*; D008297:Male; D010375:Pedigree; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D010766:Phosphorylation; D011110:Polymorphism, Genetic / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects; D014443:Tyrosine / Q000378:metabolism
D007328:Insulin; D014443:Tyrosine; D019869:Phosphatidylinositol 3-Kinases