The Adapter Protein ZIP Binds Grb14 and Regulates Its Inhibitory Action on Insulin Signaling by Recruiting Protein Kinase Cζ

Abstract

Grb14 is a member of the Grb7 family of adapters and acts as a negative regulator of insulin-mediated signaling. Here we found that the protein kinase C (PKC) interacting protein, ZIP, interacted with Grb14. Coimmunoprecipitation experiments demonstrated that ZIP bound to both Grb14 and PKC, thereby acting as a link in the assembly of a PKC-ZIP-Grb14 heterotrimeric complex. Mapping studies indicated that ZIP interacted through its ZZ zinc finger domain with the phosphorylated insulin receptor interacting region (PIR) of Grb14. PKC phosphorylated Grb14 under in vitro conditions and in CHO-IR cells as demonstrated by in vivo labeling experiments. Furthermore, Grb14 phosphorylation was increased under insulin stimulation, suggesting that the PKC-ZIP-Grb14 complex is involved in insulin signaling. The PIR of Grb14, which also interacts with the catalytic domain of the insulin receptor (IR) and inhibits its activity, was preferentially phosphorylated by PKC. Interestingly, the phosphorylation of Grb14 by PKC increased its inhibitory effect on IR tyrosine kinase activity in vitro. The role of ZIP and Grb14 in insulin signaling was further investigated in vivo in Xenopus laevis oocytes. In this model, ZIP potentiated the inhibitory action of Grb14 on insulininduced oocyte maturation. Importantly, this effect required the recruitment of PKC and the phosphorylation of Grb14, providing in vivo evidences for a regulation of Grb14-inhibitory action by ZIP and PKC. Together, these results suggest that Grb14, ZIP, and PKC participate in a new feedback pathway of insulin signaling.

The PIR domain of Grb14 is an intrinsically unstructured protein: implication in insulin signaling

“…Upon insulin stimulation, PIR binds to the activated IR regulatory kinase loop. PIR phosphorylation by PKCj via ZIP leads to an increased Grb14 inhibitory action on IR [14]. The interaction between PIR and ZIP does not alter the insulin-induced Grb14-IR interaction.…”

“…The PIR partners described so far are the IR and a new downstream partner of Grb14, ZIP [14]. A model has been proposed for the role of ZIP in the Grb14 pathway.…”

“…ZIP could act as a functional sca¡old protein connecting protein kinase Cj (PKCj) to Grb14. ZIP interacts with the V1 domain of PKCj through its AID domain and with the PIR of Grb14 through its ZZ zinc ¢nger domain [14]. Upon insulin stimulation, PIR binds to the activated IR regulatory kinase loop.…”

“…This domain is highly conserved among the members of the Grb7 family and does not show any similarity with any other known protein interaction domain. To date, only one other partner of the PIR domain has been described, the protein kinase Cj interacting protein (ZIP) [14].…”

Voies de signalisation de l’insuline : mécanismes affectés dans l’insulino-résistance

“…D'autres mécanismes sont également impliqués dans la régulation négative du signal insuline: la déphosphory-lation des phosphoinositides par des lipide-phosphatases comme PTEN (phosphatase and tensin homolog deleted on chromosome ten) et SHIP (Src homology 2 domain-containing inositol 5 -phosphatase) réversent le signal PI3 kinase. Certaines protéines sont capables de se lier au récepteur et de bloquer la transmission du signal, comme les membres de la famille GRB7, 10 et 14 (cette dernière utilisant la protéine ZIP (protein kinase C ζ interacting protein) et la PKC ζ pour rétrocontrôler le RI) [16] et les protéines de la famille SOCS (suppressor of cytokine signaling), impliquées dans la régula-tion négative de la signalisation par les cytokines. La régulation de la quantité des protéines substrats IRS qui sont dégradées après ubiquitination va également contrôler la réponse à l'insuline.…”