Role of IRS and PHIP on insulin-induced tyrosine phosphorylation and distribution of IRS proteins.
To analyze the functional differences of the insulin receptor substrate (IRS) family, the N-terminal fragments containing the pleckstrin homology (PH) domains and the phosphotyrosine-binding (PTB) domains of IRS (IRS-N) proteins, as well as intact IRS molecules, were expressed in Cos-1 cells, and insulin-induced tyrosine phosphorylation and subcellular distribution of IRS proteins were analyzed. In contrast to the distinct affinities toward phosphoinositides, these IRS-N fragments non-selectively inhibited insulin-induced tyrosine phosphorylation of IRS-1, IRS-2 and IRS-3, among which IRS3-N was most effective. The mutations of IRS-1 disrupting all the phosphoinositide-binding sites in both the PH and PTB domains significantly but not completely suppressed tyrosine phosphorylation of IRS-1, which was further inhibited by coexpression of all the IRS-N proteins examined. In contrast, the N-terminal PH domain-interacting region (PHIP-N) of PH-interacting protein (PHIP) did not impair tyrosine phosphorylation of either IRS molecule. The analysis using confocal microscopy also demonstrated that all the IRS-N proteins, but not PHIP-N, suppressed targeting of IRS-1 to the plasma membrane in response to insulin. Moreover, the phosphoinositide affinity-disrupting mutations of IRS-1 significantly impaired but did not completely abrogate the insulin-induced translocation of IRS-1 to the plasma membrane, which was further suppressed by IRS1-N overexpression. These findings suggest that both insulin-induced tyrosine phosphorylation and the cell surface targeting of IRS proteins may be regulated in a similar manner through a target molecule common to the members of the IRS family, and distinct from phosphoinositides or PHIP.
Kaburagi
Yasushi
Y
Department of Metabolic Disorder, Research Institute, International Medical Center of Japan, Toyama, Shinjuku-ku, Tokyo, Japan. kaburagi@ri.imcj.go.jp
Okochi
Hitoshi
H
Satoh
Shinobu
S
Yamashita
Ryo
R
Hamada
Keiko
K
Ikari
Kohei
K
Yamamoto-Honda
Ritsuko
R
Terauchi
Yasuo
Y
Yasuda
Kazuki
K
Noda
Mitsuhiko
M
D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D001798:Blood Proteins / Q000378:metabolism; D019556:COS Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D006801:Humans; D015151:Immunoblotting; D007328:Insulin / Q000494:pharmacology*; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D018613:Microscopy, Confocal; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010716:Phosphatidylinositols / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D014443:Tyrosine / Q000378:metabolism*
D001798:Blood Proteins; D002352:Carrier Proteins; C526292:IRS1 protein, human; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; D010716:Phosphatidylinositols; D010750:Phosphoproteins; C039077:platelet protein P47; D019000:Phosphotyrosine; D014443:Tyrosine; D011972:Receptor, Insulin