Interaction of wild type and dominant-negative p55PIK regulatory subunit of phosphatidylinositol 3-kinase with insulin-like growth factor-1 signaling proteins.
In a first series of experiments done in the yeast two-hybrid system, we investigated the nature of protein-protein interaction between the regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase), p55PIK, and several of its potential signaling partners. The region between the Src homology 2 (SH2) domains of p55PIK bound to the NH2 terminus region of p110alpha, as previously shown for p85alpha. Moreover, we found that the insulin-like growth factor-1 receptor (IGF-IR) bound to p55PIK; the interaction occurred at the receptor tyrosine 1316 and involved both p55PIK SH2 domains. Interaction between p55PIK and IGF-IR was seen not only in the yeast two-hybrid system, but also using in vitro binding and coimmunoprecipitation of lysates from IGF-1 stimulated 293 cells overexpressing p55PIK. Further, IGF-I stimulation of these cells led to tyrosine phosphorylation of p55PIK. In 293 cells association of p55PIK with insulin receptor substrate-1 and with IGF-IR was dependent on PI 3-kinase, since it was increased by wortmannin, an inhibitor of PI 3-kinase. Further, by deleting amino acids 203-217 of p55PIK inter-SH2 domain, we engineered a p55PIK mutant unable to bind to the p110alpha catalytic subunit of PI 3-kinase. This mutant had a dominant-negative action on insulin-stimulated glucose transport, since insulin's effect on Glut 4 myc translocation was inhibited in adipocytes expressing mutant p55PIK. Importantly, this dominant-negative mutant was more efficient than wild type p55PIK in associating to IGF-IR and insulin receptor substrate-1 in 293 cells. Taken together, our results show that p55PIK interacts with key elements in the IGF-I signaling pathway, and that these interactions are negatively modulated by PI 3-kinase itself, providing circuitry for regulatory feedback control.
Mothe
I
I
Institut National de la Santé et de la Recherche Médicale U145, Nice, France.
Delahaye
L
L
Filloux
C
C
Pons
S
S
White
M F
MF
Van Obberghen
E
E
D001665:Binding Sites; D001692:Biological Transport / Q000187:drug effects; D005656:Fungal Proteins / Q000378:metabolism; D017930:Genes, Reporter; D005947:Glucose / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology; D055504:Insulin Receptor Substrate Proteins; D007334:Insulin-Like Growth Factor I / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D017526:Receptor, IGF Type 1 / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D015398:Signal Transduction / Q000235:genetics*
D005656:Fungal Proteins; D005947:Glucose; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D007334:Insulin-Like Growth Factor I; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins; D017526:Receptor, IGF Type 1; D011993:Recombinant Fusion Proteins