In this study, we identified a novel interaction between PLC-γ1 and Akt. We showed by immunofluorescence and subcellular fractionation that PLC-γ1 and Akt were colocalized to the plasma membrane after EGF stimulation (). We also showed by immunoprecipitation, GST-fusion protein pull-down experiments, and in vitro binding experiments that PLC-γ1 and Akt directly bind to each other (Figures and ). It is well established that activated EGFR directly stimulates PLC-γ1 by phosphorylating its tyrosine residues and indirectly stimulates Akt by activating PI3K. Both PLC-γ1 and PI3K–Akt pathways play significant roles in EGFR-mediated cell signaling. Although both PLC-γ1 and PI3K-Akt play distinct role in cell signaling, the two pathways have been shown to interact each other. For example, PLC-γ1 and PI3K are both involved in lipid metabolism and use the same substrate (PIP2
) (Carpenter and Ji, 1999
). Both PLC-γ1 and PI3K–Akt pathways are involved in cell mitogenesis and motility (Chen et al., 1994
). It is known that PLC-γ1 may regulate Ras activity through the activation of rasGRP, and activated Ras then stimulates the activation of PI3K (Rodriguez-Viciana et al., 1994
; Ehrhardt et al., 2004
). We recently showed that PI3K might affect the translocation of PLC-γ1 by generating phosphatidylinositol 3-phosphate (Wang and Wang, 2003
). So far, the data regarding the interaction between PLC-γ1 pathway and PI3K–Akt pathway are exclusively between PLC-γ1 and PI3K. Here, we demonstrate that PLC-γ1 and Akt directly bind each other, which provides another layer of interaction between PLC-γ1 pathway and PI3K–Akt pathway.
We further showed that the interaction between PLC-γ1 and Akt are mediated by PLC-γ1 SH3 domain () and the proline-rich motifs of Akt (). PLC-γ1 is a lipase with multiple domains, which allows the interaction of PLC-γ1 with various proteins to form large protein complexes. It is clear that PLC-γ1 SH2 domains interact with the phosphotyrosine of RTKs. However, the functions of PLC-γ1 SH3 and PH domains are still not clear. Interestingly, most newly identified interactions between PLC-γ1 and other proteins are mediated by its SH3 domains. It was recently reported that PLC-γ1 directly interact with various cellular proteins, including PLD2
(Jang et al., 2003
), PIKE (Ye et al., 2002
), dynamin (Choi et al., 2004
), Emt (Perez-Villar and Kanner, 1999
), and SOS (Kim et al., 2000
) through its SH3 domain. Our studies demonstrate that Akt can be added to this list.
Akt contains two proline-rich motifs (424PFKP427 and 467PHFP470) within its C terminus. We showed that point mutation of either P424 or P467 to alanine significantly reduced but not abolish the interaction between PLC-γ1 and Akt, whereas a mutant Akt with both proline mutated to alanine (P/P) did not bind to PLC-γ1 at all (). This suggests that both proline motifs of Akt are involved in the interaction with PLC-γ1.
Different from the interaction between SH2 domain and phosphotyrosine motif, the interaction between SH3 domain and proline-rich motifs does not require the posttranslational modification of either SH3 domain or the proline motifs. Indeed, we showed here by in vitro binding assay that the interaction between PLC-γ1 SH3 domain and Akt is constitutive (). However, these data seem at odds with our in vivo data showing that the interaction between the full-length PLC-γ1 and Akt is dependent on EGF stimulation. Similar observation has been made for the interactions between PLC-γ1 and other proteins, such as PLD2
(Jang et al., 2003
), PIKE (Ye et al., 2002
), dynamin (Choi et al., 2004
), and Emt (Perez-Villar and Kanner, 1999
In this study, we provide a novel model to address this discrepancy. We proposed that in vivo, the full-length PLC-γ1 adopted a conformation that restricts the interaction of its SH3 domain with Akt. Phosphorylation of PLC-γ1 Y771 and/or Y783 by EGF stimulation releases the restriction on SH3 domain (). Our model is supported by the following evidence. First, Y771 and Y783 are the major tyrosine residues phosphorylated by EGFR and located between PLC-γ1 SH3 and CSH2 domains. Thus, it is highly possible that the phosphorylation of Y771 and/or Y783 may have major effects on the conformation of PLC-γ1. Second, it was recently reported that in response to EGF, the phosphorylated Y783 forms an intramolecular interaction with the CSH2 domain in the activated PLC-γ1 (Poulin et al., 2005
). Third, we showed in this study that double mutation of Y771 and Y783 or single mutation of Y783 blocked EGF-induced association between PLC-γ1 and Akt ().
Figure 9. Model to illustrate EGF-induced interaction between Akt and PLC-γ1. Before EGF stimulation, both PLC-γ1 and Akt are located in the cytosol. The conformation of inactive PLC-γ1 blocks the interaction between PLC-γ1 SH3 domain (more ...)
We also examined the role of PLC-γ1–Akt interaction in EGF-induced cell signaling. Akt is a serine/threonine kinase. The minimal substrate consensus sequence for Akt is RXRXXS/T. Interestingly, the amino acid sequence leading to S1248 is RAREGS, which suggests that S1248 could be phosphorylated by Akt. Indeed, we showed that the interaction between PLC-γ1 and Akt resulted in the phosphorylation of PLC-γ1 S1248 by Akt (). Inhibition of Akt phosphorylation by wortmannin or disruption of PLC-γ1 and Akt interaction by mutation partially blocked EGF-induced phosphorylation of PLC-γ1 S1248 (), which suggests that EGF-induced PLC-γ1 S1248 phosphorylation is partially dependent on binding to Akt. Because it has been suggested that PKA could phosphorylate PLC-γ1 S1248 (Park et al., 1992
), we further examined the relative contribution of Akt and PKA on EGF-induced PLC-γ1 S1248 phosphorylation. Our results indicated that both Akt and PKA partially contributed to EGF-induced PLC-γ1 S1248 phosphorylation ().
Finally, we showed that the interaction between PLC-γ1 and Akt plays important role in EGF-induced cell motility. Both PLC-γ1 and Akt have been shown to regulate EGF-induced cell motility (Chen et al., 1994
). We showed here that the enhancement of EGF-induced cell motility by PLC-γ1 is mostly abolished by mutation of its S1248 (). This indicates that S1248 phosphorylation by Akt is important in mediating EGF-induced cell motility. We further showed that the enhancement of cell motility by Akt is abolished by blocking the interaction between Akt and PLC-γ1 and by inhibiting PLC-γ1 activity (). All these data support an important role for Akt–PLC-γ1 interaction in EGF-induced cell motility. It was reported that inhibitors of PKC, which inhibit serine phosphorylation of PLC-γ1, also inhibit the tyrosine phosphorylation and catalytic activity PLC-γ1 (Yamada et al., 1992
). Wortmannin, an inhibitor of PI3K, which inhibits Akt indirectly, also inhibits the catalytic activity of PLC-γ1. PLC-γ1 catalytic activity is involved in the EGF-mediated cell motility (Chen et al., 1994
). Therefore, Akt may regulate the EGF-mediated cell motility through regulating the catalytic activity of PLC-γ1.
Whether the interaction between PLC-γ1 and Akt will also modulate Akt activity and function is still not clear. It was reported that PLC-γ2, a homologue of PLC-γ1, is required for B cell receptor-mediated cell survival (Bell et al., 2004
). Akt activation is abolished by the PLC-γ1 inhibitor U73122, which suggests a possible role of PLC-γ1 in EGF-induced cell survival through the interaction with Akt (Deb et al., 2004
). Therefore, whether the interaction between PLC-γ1 and Akt regulates cell survival warrants future research.
In summary, we identified a novel interaction between PLC-γ1 and Akt. We demonstrated that the interaction is mediated by the binding of PLC-γ1 SH3 domain to Akt proline-rich motifs. Both Akt proline-rich motifs interact with PLC-γ1 SH3 domain. We also provide a novel model to depict how the interaction between PLC-γ1 SH3 domain and Akt proline motifs is dependent on EGF stimulation (). In this model, the SH3 domain of inactive PLC-γ1 is not accessible to the proline motifs of Akt. Phosphorylation of Y783 by EGFR changed PLC-γ1 conformation by forming an intramolecular association between Y783 and CSH2 domain in PLC-γ1. This conformation change results in the interaction between PLC-γ1 SH3 domain and Akt proline motifs. Furthermore, we showed that the interaction between PLC-γ1 and Akt caused phosphorylation of PLC-γ1 S1248 and plays important role in EGF-stimulated cell motility.