Interaction with Grb14 results in site-specific regulation of tyrosine phosphorylation of the insulin receptor.
The dynamics of interaction of the insulin receptor (IR) with Grb14 was monitored, in real time, in living human embryonic kidney cells, using bioluminescence resonance energy transfer (BRET). We observed that insulin rapidly and dose-dependently stimulated this interaction. We also observed that insulin-induced BRET between the IR and protein tyrosine phosphatase 1B (PTP1B) was markedly reduced by Grb14, suggesting that Grb14 regulated this interaction in living cells. Using site-specific antibodies against phosphorylated tyrosines of the IR, we showed that Grb14 protected the three tyrosines of the kinase loop from dephosphorylation by PTP1B, while favouring dephosphorylation of tyrosine 972. This resulted in decreased IRS-1 binding to the IR and decreased activation of the extracellular signal-regulated kinase pathway. Increased Grb14 expression in human liver-derived HuH7 cells also seemed to specifically decrease the phosphorylation of Y972. Our work therefore suggests that Grb14 may regulate signalling through the IR by controlling its tyrosine dephosphorylation in a site-specific manner.
Département de Biologie Cellulaire, Institut Cochin, Université Paris Descartes, 22 Rue Méchain, UMRCNRS 8104, INSERM U567, Paris 75014, France.
Sébastien
S
Nouaille
Christophe
C
Blanquart
Vladimir
V
Zilberfarb
Nicolas
N
Boute
Dominique
D
Perdereau
Johan
J
Roix
Anne-Françoise
AF
Burnol
Tarik
T
Issad
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D006801:Humans; D049449:Luminescence; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D025941:Protein Interaction Mapping; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1 / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011972:Receptor, Insulin / Q000378:metabolism* / Q000502:physiology; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology; D014443:Tyrosine / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D001426:Bacterial Proteins; C099655:GRB14 protein, human; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D014443:Tyrosine; D011972:Receptor, Insulin; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1