Spatial and temporal aspects and the interplay of Grb14 and protein tyrosine phosphatase-1B on the insulin receptor phosphorylation

Cell Communication and Signaling Spatial and temporal aspects and the interplay of Grb14 and protein tyrosine phosphatase-1B on the insulin receptor phosphorylation Raju VS Rajala 0 1 Devaraj K Basavarajappa 1 Radhika Dighe 1 Ammaji Rajala 1 0 Departments of Physiology, University of Oklahoma Health Sciences Center , Oklahoma City, OK 73104 , USA 1 Departments of Ophthalmology, University of Oklahoma Health Sciences Center , Oklahoma City, OK 73104 , USA Background: Growth factor receptor-bound protein 14 (Grb14) is an adapter protein implicated in receptor tyrosine kinase signaling. Grb14 knockout studies highlight both the positive and negative roles of Grb14 in receptor tyrosine kinase signaling, in a tissue specific manner. Retinal cells are post-mitotic tissue, and insulin receptor (IR) activation is essential for retinal neuron survival. Retinal cells express protein tyrosine phosphatase-1B (PTP1B), which dephosphorylates IR and Grb14, a pseudosubstrate inhibitor of IR. This project asks the following major question: in retinal neurons, how does the IR overcome inactivation by PTP1B and Grb14? Results: Our previous studies suggest that ablation of Grb14 results in decreased IR activation, due to increased PTP1B activity. Our research propounds that phosphorylation in the BPS region of Grb14 inhibits PTP1B activity, thereby promoting IR activation. We propose a model in which phosphorylation of the BPS region of Grb14 is the key element in promoting IR activation, and failure to undergo phosphorylation on Grb14 leads to both PTP1B and Grb14 exerting their negative roles in IR. Consistent with this hypothesis, we found decreased phosphorylation of Grb14 in diabetic type 1 Ins2Akita mouse retinas. Decreased retinal IR activation has previously been reported in this mouse line. Conclusions: Our results suggest that phosphorylation status of the BPS region of Grb14 determines the positive or negative role it will play in IR signaling. Grb14; PTP1B; Shp2; SRC activation; Tyrosine phosphorylation; Insulin receptor; Tyrosine kinase signaling - Introduction Growth factor receptor-bound protein 14 (Grb14) is an adaptor protein that is known to interact with a number of receptor tyrosine kinases and signaling molecules [1,2]. Grb14 has an inhibitory effect on receptor tyrosine kinase signaling and, in particular, on insulin receptor signaling [3]. Consistent with these findings, a genome-wide association study demonstrated that single nucleotide polymorphisms at Grb14 are strongly associated with reduced insulin sensitivity in diabetic patients [4]. While there is convincing evidence of a negative role of Grb14 in insulin signaling [5,6], experiments with Grb14-/- animals have also revealed positive effects of Grb14 on receptor tyrosine kinase signaling, in a tissue specific manner [7,8]. We previously identified Grb14 in retinal tissues [9]. Interestingly, Grb14 undergoes a light-dependent intracellular translocation within rod photoreceptor neurons [8]. Light induces activation of the insulin receptor (IR) and ablation of Grb14 results in the loss of light-dependent activation of the IR [8]. In photoreceptors, Grb14 undergoes tyrosine phosphorylation by light-activated non-receptor tyrosine kinase Src, and phosphorylated Grb14 (Grb14-P) acts as a positive regulator of the IR by inhibiting PTP1B, a negative regulator of the IR [10]. Very recently, we reported that Grb14 modulates the activity of the rod cyclic nucleotide gated channel (CNG), and perhaps cGMP-phospho diesterase in regulating rod transduction and light adaptation [11]. We also revealed that CNG channel phosphorylation is regulated by IR [12], while Grb14 regulates both IR activation and CNG channel modulation [10,11,13]. A high expression of Grb14 in myocardial tissue activates the PI3K-Akt pathway: ablation of Grb14 results in myocardial infarction and decreased PI3K/Akt activation [7]. In several models of insulin resistance, increased expression of Grb14 in adipose tissue has previously been reported [14]. Convincing evidence for a negative role of Grb14 in insulin signaling exists [15]. This evidence shows enhanced glucose tolerance and insulin sensitivity in Grb14-deficient mice [5]. Thus, our primary research question is how Grb14 achieves negative or positive roles in IR signaling. The molecular switch determining whether Grb14 will perform a particular role is unknown. Our studies suggest that the phosphorylation status of Grb14 is the key element in determining whether it will execute a negative or positive role in IR signaling. Results Effect of a phosphorylated BPS region of Grb14 on IR kinase activity To determine whether Grb14 phosphorylation performs any role in IR kinase activity, we examined the effect of non-phosphorylated and phosphorylated BPS regions of Grb14 on IR kinase activity in vitro (Figure 1A). Nonphosphorylated and phosphorylated BPS domains of GSTGrb14 were expressed alone, or co-expressed with VSRC and purified according to the method described earlier [10]. Both inhibited the IR kinase activity equally well (Figure 1B). The crystal structure of the BPS domain revealed that a region between amino acids 373 and 381 is involved in binding to the IR, and that Glu373 is crucial for this binding [16]. The mutant GST-BPS-SH2 (E373Q) and wild-type GST-BPS-SH2 Grb14 were either expressed alone or coexpressed with VSRC [17]. The expressed proteins were purified and immunoblotted with anti-PY99 antibody. The results indicated that VSRC mediated phosphorylation of the BPS-SH2 (E373Q) and wild-type BPS-SH2 domains (Figure 1C). To ensure equal amounts of fusion proteins, we reprobed the blot with anti-GST antibody (Figure 1C). Non-phosphorylated and phosphorylated BPS-SH2 (E3 73Q) domains of Grb14 were tested for their effect on IR kinase activity. This mutant BPS region failed to inhibit the IR kinase activity, regardless of phosphorylation status (Figure 1B). However, the same mutant was able to inhibit PTP1B activity, similar to the wild-type BPS region of Grb14, and the inhibition caused by the phosphorylated E373Q-BPS region was significantly greater than that of the non-phosphorylated E373Q-BPS-SH2 domain (Figure 1D). These results show that phosphorylated and non-phosphorylated BPS domains equally inhibit IR kinase activity, whereas the phosphorylated BPS domain significantly inhibits PTP1B activity to a greater extent than its non-phosphorylated counterpart. These data suggest that IR and PTP1B inhibitory activities are influenced by the BPS region of Grb14. Interaction of phosphorylated Grb14 with vSrc-SH2 domain The Tyr347 residue in the BPS region of Grb14 is essential for PTP1B binding and inhibition of its activity [10]. The SH2 domain of the Src family recognizes the preferred sequence with the general motif pTyr-hydrophilichydrophilic-Ile/Pro [18]. The phosphorylated Grb14 BPS region has the sequence pTyr-Gln-Asn-Tyr followed by a bulky side chain of Met, which (...truncated)