Rajala et al. Cell Communication and Signaling 2013, 11:96 http://www.biosignaling.com/content/11/1/96 RESEARCH Open Access Spatial and temporal aspects and the interplay of Grb14 and protein tyrosine phosphatase-1B on the insulin receptor phosphorylation Raju VS Rajala1,2,3,4*, Devaraj K Basavarajappa1,4,5, Radhika Dighe1,4 and Ammaji Rajala1,4 Abstract 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. Keywords: Grb14, PTP1B, Shp2, SRC activation, Tyrosine phosphorylation, Insulin receptor, Tyrosine kinase signaling Introduction revealed positive effects of Grb14 on receptor tyrosine kin- Growth factor receptor-bound protein 14 (Grb14) is an ase signaling, in a tissue specific manner [7,8]. adaptor protein that is known to interact with a number We previously identified Grb14 in retinal tissues [9]. of receptor tyrosine kinases and signaling molecules Interestingly, Grb14 undergoes a light-dependent intra- [1,2]. Grb14 has an inhibitory effect on receptor tyrosine cellular translocation within rod photoreceptor neurons kinase signaling and, in particular, on insulin receptor sig- [8]. Light induces activation of the insulin receptor (IR) and naling [3]. Consistent with these findings, a genome-wide ablation of Grb14 results in the loss of light-dependent acti- association study demonstrated that single nucleotide poly- vation of the IR [8]. In photoreceptors, Grb14 undergoes morphisms at Grb14 are strongly associated with reduced tyrosine phosphorylation by light-activated non-receptor insulin sensitivity in diabetic patients [4]. While there is tyrosine kinase Src, and phosphorylated Grb14 (Grb14-P) convincing evidence of a negative role of Grb14 in insulin acts as a positive regulator of the IR by inhibiting PTP1B, a -/- signaling [5,6], experiments with Grb14 animals have also negative regulator of the IR [10]. Very recently, we reported that Grb14 modulates the activity of the rod cyclic nucleo- tide gated channel (CNG), and perhaps cGMP-phospho * Correspondence: [email protected] diesterase in regulating rod transduction and light adapta- 1Departments of Ophthalmology, University of Oklahoma Health Sciences tion [11]. We also revealed that CNG channel phosphoryl- Center, Oklahoma City, OK 73104, USA ation is regulated by IR [12], while Grb14 regulates both 2Departments of Physiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA IR activation and CNG channel modulation [10,11,13]. Full list of author information is available at the end of the article A high expression of Grb14 in myocardial tissue activates © 2013 Rajala et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Rajala et al. Cell Communication and Signaling 2013, 11:96 Page 2 of 11 http://www.biosignaling.com/content/11/1/96 the PI3K-Akt pathway: ablation of Grb14 results in myo- data suggest that IR and PTP1B inhibitory activities are cardial infarction and decreased PI3K/Akt activation [7]. In influenced by the BPS region of Grb14. several models of insulin resistance, increased expression of Grb14 in adipose tissue has previously been reported Interaction of phosphorylated Grb14 with vSrc-SH2 domain [14].ConvincingevidenceforanegativeroleofGrb14in The Tyr347 residue in the BPS region of Grb14 is es- insulin signaling exists [15]. This evidence shows enhanced sential for PTP1B binding and inhibition of its activity glucose tolerance and insulin sensitivity in Grb14-deficient [10]. The SH2 domain of the Src family recognizes the pre- mice [5]. Thus, our primary research question is how ferred sequence with the general motif pTyr-hydrophilic- Grb14 achieves negative or positive roles in IR signaling. hydrophilic-Ile/Pro [18]. The phosphorylated Grb14 BPS The molecular switch determining whether Grb14 will per- region has the sequence pTyr-Gln-Asn-Tyr followed by a form a particular role is unknown. Our studies suggest that bulky side chain of Met, which is similar to the preferred the phosphorylation status of Grb14 is the key element in interacting sequences of the SH2 domain of Src. To in- determining whether it will execute a negative or positive vestigate whether the SH2 domain of vSrc binds to the role in IR signaling. phosphorylated BPS region, the expressed His-tagged BPS region of Grb14, either alone or co-expressed with VSRC in E. coli (Figure 2A), was subjected to a GST pull-down Results assay with either GST or GST-vSrc-SH2 fusion proteins. Effect of a phosphorylated BPS region of Grb14 on IR The bound proteins were immunoblotted with anti-His kinase activity antibody. The vSrc-SH2 domain associated with the BPS To determine whether Grb14 phosphorylation performs region only under phosphorylated conditions (Figure 2B). any role in IR kinase activity, we examined the effect of The blot was reprobed with anti-GST antibody to en- non-phosphorylated and phosphorylated BPS regions of sure equal amounts of fusion proteins in each pull-down Grb14 on IR kinase activity in vitro (Figure 1A). Non- (Figure 2C-D). phosphorylated and phosphorylated BPS domains of GST- We previously reported a light-dependent phosphoryl- Grb14 were expressed alone, or co-expressed with VSRC ation of Grb14 in vivo [10]. In this study, we examined and purified according to the method described earlier whether the vSrc-SH2 domain is able to bring down phos- [10]. Both inhibited the IR kinase activity equally well phorylated Grb14. We subjected the dark- and light- (Figure 1B). The crystal structure of the BPS domain adapted mouse retina lysates [19] of wild-type and Grb14-/- revealed that a region between amino acids 373 and 381 is mice (Figure 2H) to a GST-vSrc-SH2 pull-down assay. The involved in binding to the IR, and that Glu373 is crucial bound proteins underwent immunoblotting with anti-PY99 for this binding [16]. and anti-Grb14 antibodies (Figure 2E-F). To ensure equal The mutant GST-BPS-SH2 (E373Q) and wild-type amounts of fusion proteins, we reprobed the blot with anti- GST-BPS-SH2 Grb14 were either expressed alone or co- GST antibody (Figure 2G). The results revealed the pres- expressed with VSRC [17]. The expressed proteins were ence of phosphorylation on Grb14 and its association with purified and immunoblotted with anti-PY99 antibody. the vSrc-SH2 domain, specifically in light-adapted wild-type The results indicated that VSRC mediated phosphoryl- retinas, but not in dark-adapted wild-type retinas or in ation of the BPS-SH2 (E373Q) and wild-type BPS-SH2 dark- and light-adapted Grb14-/- mice (Figure 2E-F). These domains (Figure 1C). To ensure equal amounts of fusion data suggest that Grb14 undergoes light-dependent phos- proteins, we reprobed the blot with anti-GST antibody phorylation in the retina, and that the vSrc-SH2 domain is (Figure 1C). able to associate with phosphorylated Grb14 in vitro. This Non-phosphorylated and phosphorylated BPS-SH2 (E3 also indicates the specificity of the interaction between 73Q) domains of Grb14 were tested for their effect on Grb14 and the vSrc-SH2 domain in vitro. IR kinase activity. This mutant BPS region failed to in- We previously reported that Grb14 phosphorylation hibit the IR kinase activity, regardless of phosphorylation was absent in the retinal pigment epithelium 65 knockout status (Figure 1B). However, the same mutant was able (Rpe65-/-) mice [10]. Rpe65-/- mice have opsin in their to inhibit PTP1B activity, similar to the wild-type BPS photoreceptor outer segments, but do not form photo- region of Grb14, and the inhibition caused by the phos- bleachable rhodopsin (the activated form), due to the ab- phorylated E373Q-BPS region was significantly greater sence of regeneration of the chromophore 11-cis-retinal than that of the non-phosphorylated E373Q-BPS-SH2 [20]. To further confirm the specificity of the vSrc-SH2 domain (Figure 1D). These results show that phosphory- domain with phosphorylated Grb14, we subjected the ret- lated and non-phosphorylated BPS domains equally in- inal lysates of the wild-type and Rpe65-/- mice to GST- hibit IR kinase activity, whereas the phosphorylated BPS vSrc-SH2 pull-down and immunoblotting with anti-PY99 domain significantly inhibits PTP1B activity to a greater (Figure 2I) and anti-Grb14 (Figure 2J) antibodies. To en- extent than its non-phosphorylated counterpart.