Although the Grb family of proteins was described for the first time over a decade ago, still much has to be learned about the role of these adaptors in various signaling pathways. Most of what we know about these proteins has come from binding studies and elucidation of the various domains involved in these interactions. Grb7, 10 and 14 have been shown to bind a number of receptors and intracellular proteins primarily via SH2 mediated interactions, although the importance of the BPS domain is also becoming apparent.
Grb14 was previously identified in a yeast two-hybrid screen as a binding partner for the endothelial receptor Tie2 [5
]. Tie2 is an angiogenic RTK involved in numerous aspects of endothelial biology such as cell migration, cell survival and tubule formation. We have now been able to show that Grb14 is endogenous to endothelial cells further supporting a role for Grb14 in endothelial cell signaling. Furthermore, the results reported herein are the first to describe tyrosine phosphorylation of Grb14. This phosphorylation appears to require a kinase competent Tie2 and tyrosines 1100 and 1106 on the receptor.
Phosphorylation is the most widespread post-translational protein modification in cell signaling [17
]. Phosphorylation of a protein can modulate its behavior in a multitude of ways including its function, localization, half life and binding to other molecules [18
]. In eukaryotes, phosphorylation is typically 'carried out' on serine, threonine or tyrosine residues.
Since there discovery, Grb family members have been shown become phosphorylated under a number of various conditions. The role of this phosphorylation, however, remains somewhat enigmatic. All family memebers (Grb7, 10 and 14) have been shown to possess both basal and growth factor induced serine phosphorylation (reviewed in Holt, 2005 [19
]). Grb10 and Grb7 have also been shown to be tyrosine phosphorylated. More specifically, Grb10 and Grb7 tyrosine phosphorylation has been previously implicated in signaling downstream of endothelial receptors. Grb10 has been shown to be tyrosine phosphorylated in endothelial cells by VEGF [20
]. Our lab has also previously shown that Grb7 becomes tyrosine phosphorylated in the presence of the endothelial receptor Tie2 [5
]. Taken together, these data supports a role for Grb proteins in endothelial biology.
The current study suggests that Grb14 tyrosine phosphorylation requires a kinase competent Tie2 receptor. In a separate experiment, Grb14 was not tyrosine phosphorylated in the presence of EGFR suggesting some specificity for the Tie2 kinase (data not shown). Whether or not Grb14 is a direct substrate of the Tie2 kinase activity, or rather is phosphorylated by an alternate kinase recruited to this receptor, remains to be seen.
Grb proteins do not possess intrinsic kinase activity, but have been shown to bind a number of receptor and non-receptor kinases (reviewed in [7
]. In insulin signaling, Grb10 serine phosphorylation appears to involve the Pi3K and MAPK signaling pathways, while PKCζ seems to play a role in Grb14 phosphorylation [11
]. Grb proteins have been found to bind a number of tyrosine kinases (reviewed in Holt and Siddle, 2005 [19
]). Interestingly, in the case of insulin signaling, although Grb10 and Grb14 bind the IR, they do not appear to be direct substrates for the IR tyrosine kinase activity [13
]. Instead, at least with respect to Grb10, Src/Fyn kinases were suggested to be responsible. In general, how and why Grb proteins are phosphorylated remains to be determined in most cases.
In our studies, Grb14 tyrosine phosphorylation was abolished upon mutation of Y1100 and Y1106 to phenylalanine in the receptor double mutant Tie21100/1106, suggesting these two residues may play a role in Grb14 tyrosine phosphorylation downstream of Tie2. This should perhaps not be surprising given that there is emerging evidence that the Grb family members may bind their target proteins via two separate regions, the SH2 and BPS domains (Reviewed in Holt and Siddle, 2005 [19
This is particularly evident in the case of insulin signaling where both SH2 and BPS domains of Grb 7, 10 and 14 have been shown to bind the IR [19
]. Specifically, structural studies have shown that the Grb14 SH2 and BPS domains bind phosphorylated tyrosine residues within the IR activation loop [24
]. Whether tyrosines 1100 and 1106 bind Grb14 directly or play a more indirect role in Grb14 tyrosine phosphorylation, however, remains to be determined.
It is interesting to note that in comparison with the other Grb family members, Grb14 has been shown to interact with a relatively small number of receptors. In accordance with this observation, we have found in vivo
binding studies of Grb14 and Tie2 have been particularly challenging (data not shown). This may be explained, at least in part, by structural studies which have shown that Grb14 may have more difficulty binding to phosphotyrosine containing ligands due to the presence of a non-glycyl residue at the end of the BC loop and the lack of a P+3 binding pocket in the SH2 domain [25
Further analysis of Grb14 tyrosine phosphorylation will no doubt provide information that may help elucidate the role of this protein in Tie2 signaling. Mapping the tyrosines on Grb14 which become tyrosine phosphorylated may give us a clue as to what other proteins bind and are involved in Grb14 signaling. Tyr67 on Grb10 was identified as major site of phosphorylation in response to insulin signaling. However, this site is not conserved in Grb7 and 14. Interestingly, mutation of this site increased affinity of Grb10 for IR. This raises the possibility that tyrosine phosphorylation may be involved in terminating Grb signaling at the receptor level. Alternatively, it may suggest that Grb tyrosine phosphorylation recruits this adaptor for involvement in non-receptor mediated signaling pathways. Further understanding of these sorts of post-translational modifications seen in the Grb family will no doubt shed considerable insight into their biological role.