It has recently become apparent that PLD activity plays a significant role in transduction of intracellular signals (11
). Since tyrosine kinases have been shown to form networks with a diverse array of cellular signal transduction pathways, a PLD-tyrosine kinase link provides novel mechanisms for PLD signaling to modulate a broad range of physiological responses. Therefore, it is crucial to understand how these two signaling components communicate with each other.
Several studies indicate that PTKs participate in the PLD activation via an unidentified mechanism (38
). However, the biological significance of phosphorylation as one of the activation mechanisms of PLD still remains to be determined, since PLD activity has not been correlated with its phosphorylation level in many studies (18
). In addition, the nature of the tyrosine kinase has not yet been defined. Recent studies demonstrated that PLD1 and PLD2 were phosphorylated in cell-free preparations by p38MAP kinase (39
) but such phosphorylation did not enhance PLD activity. It was suggested that an ERK-dependent tyrosine kinase might mediate the effect of norepinephrine on tyrosine phosphorylation of PLD2 (41
). Although PLD is phosphorylated on serine and threonine residues in intact cells in response to phorbol myristate acetate (25
) or on tyrosine residues by pervanadate (31
), it is still unclear whether this phosphorylation of PLD is of physiological relevance to enhanced hydrolysis of phosphatidylcholine or other phospholipid substrates.
Although Src tyrosine kinase has been shown to play a role in PLD-mediated signaling (23
), the link between c-Src and PLD was not clear. In this study, we demonstrate for the first time that Src family protein kinases induced tyrosine phosphorylation of PLD isozymes, most notably PLD2, by transient transfection. Moreover, EGF induced tyrosine phosphorylation of PLD2 in A431 cells. However, EGF-induced PLD activity was not altered by coexpression of c-Src in COS-7 cells. In addition, EGF-induced PLD activity in hepatoma cells overexpressing constitutive active mutant c-Src was not different from the activity shown in cells transfected with empty vector or dominant inactive c-Src. These results suggest that tyrosine phosphorylation of PLD by c-Src does not increase PLD phospholipase activity. This correlates with a previous report that overexpression of active c-Src or kinase-negative mutant c-Src did not affect PLD activity in rat mesangial cells (44
) but is in contrast to observations with fibroblasts transformed by the viral counterpart and oncogenic PTK v-Src (20
). A comparison of the effects of pp60c-src
would suggest that only the oncogenic tyrosine kinase is able to interfere with the Ras pathway. The significance of PLD activation by v-src tyrosine kinase is unknown. Moreover, the molecular basis by which PLD activity is differentially regulated by v-Src and c-Src remains to be clarified. In some pathways, the enzymatic activity of PLD as a lipid hydrolytic enzyme might not be strictly required for PLD function, pointing to the multifaceted nature of the PLD protein. Consistent with this hypothesis, it was reported that the mitogenic activity of phospholipase C-γ1, which is known to play an important role in cell proliferation, does not exclusively result from the enzymatic activity of the lipase and that another activity inherent to the phospholipase C-γ1 molecule can also induce DNA synthesis in quiescent cells (50
). Although it is possible that nonphospholipase activities of PLD through tyrosine phosphorylation play a role in PLD-mediated cellular signaling, at present it is still not clear how tyrosine phosphorylation of PLD contributes to PLD-mediated signal transduction in cells. Tyrosine phosphorylation of PLD may not be critical for proliferation because PLD1 and PLD2 show enhancement in a similar potency proliferation by coexpression of c-Src synergistically, even though the extent of tyrosine phosphorylation of PLD1 is very different from that of PLD2. We cannot exclude the possibility that tyrosine-phosphorylated residues in PLD2 by c-Src may provide the docking sites to recruit the proteins with the SH2 domain, leading to amplification of Src signaling cascades. The biological significance of PLD tyrosine phosphorylation remains to be determined in detail.
PLD associated with c-Src, and it was the catalytic domain, not the SH3 and SH2 domains, of c-Src that was required for binding to PLD2. This is reminiscent of the interaction of Gαs and Gαi with the catalytic domain of c-Src (30
). c-Src binds to a region of PLD2 containing PH, which is known to be involved in protein-protein interactions as well as binding of phospholipids (56
). Highly tyrosine-phosphorylated PLD2 was strongly associated with c-Src in cotransfection experiments. This was comparable to EGF-induced association of phosphorylated PLD2 with c-Src in A431 cells.
To understand PLD signaling and its biological functions, it is essential to identify the direct targets of PLD. The effectors of PLD might be proteins interacting with it or the signaling molecules that are activated by PA. Therefore, we investigated the possibility that PLD has a role not only as a substrate of c-Src but also as a regulator of c-Src. Coexpression of wild-type PLD1 or PLD2, but not catalytically inactive PLD mutants, along with c-Src, stimulated c-Src kinase activity. There seems to be little difference between PLD1 and PLD2 in c-Src activation, even though the extent of their interaction with c-Src is very different. Although catalytically inactive PLD mutant associated with c-Src, overexpression of the PLD mutant rather decreased c-Src activation. Therefore, c-Src activation via PLD appeared to be dependent upon the catalytic activity of PLD rather than interaction of PLD with c-Src. The present work identifies PLD as a regulator of Src family tyrosine kinases and provides substantial new insights for future investigations in the two fields of PLD function and Src regulators.
Until now, PLDs have not been studied for their ability to increase c-Src kinase activity. In the present study, we demonstrate for the first time that there is an intimate cross-regulation between PLD and c-Src. Critical downstream targets of the Src kinase remain controversial and probably depend on the particular signaling pathway that activated the kinase. Many proposed substrates of c-Src are derived from studies of proteins phosphorylated by the activated viral Src protein, such as phosphatidylinositol 3 kinase, Shc, phospholipase C-γ, paxillin, vinculin, and ERK (5
). In the present study, we demonstrated that PLD-induced activation of c-Src increased tyrosine phosphorylation of paxillin and activation of ERK, downstream target molecules of Src. Activation of these molecules was also dependent on the catalytic activity of PLD. Our observations are consistent with the recent report demonstrating that EGF-induced ERK activation is dependent upon PLD activity in A431 cells (46
). This may occur by stimulation of c-Src signaling via PLD activity-dependent mechanism. Therefore, our results suggested that PA generated by PLD activity might be involved in the c-Src/ERK pathway. A number of studies have demonstrated that PA induces tyrosine phosphorylation in neutrophils and other cell types (45
). While it is tempting to speculate that PA-dependent tyrosine phosphorylation may be involved in activation of c-Src and stimulation of the ERK, additional experiments are required to prove this hypothesis.
Both PLD and c-Src are overexpressed and highly activated in a wide variety of human cancers (19
). A connection between activation of Src via PLD and cancer progression would be a significant finding. Consistent with such a connection, Joseph et al. (23
) reported that elevated expression of either PLD1 or PLD2 transformed cells overexpressing c-Src. Therefore, it is reasonable to propose that these two proteins have coordinated functions.
The present studies significantly advance our understanding of the cross talk between PLDs and c-Src tyrosine kinases. In summary, our novel findings show that there is an interaction between Src and PLD protein leading to tyrosine phosphorylation of both PLD1 and PLD2 and that this interaction and PLD activity activate c-Src, leading to autophosphorylation, paxillin phosphorylation, and ERK activation. PLD is tyrosine phosphorylated as a novel substrate of c-Src and stimulates Src kinase signaling in a PLD activity-dependent mechanism, inducing cell proliferation (Fig. ). These characteristics suggest that PLD lies at an interesting nexus of Src-dependent signaling. Further studies aimed at the elucidation of cell growth signal → c-Src → signaling event of PLD phosphorylation and PLD-stimulated c-Src downstream signaling events leading to the gene expression are necessary to gain a better understanding of the functional role of PLD in mediating signals from c-Src tyrosine kinase.
FIG. 10. Proposed model of PLD-stimulated c-Src downstream signaling. EGF binds to its receptor, thereby stimulating both PLD and c-Src activation. It is likely that the resulting PLD activity is involved in the stimulation of Src-mediated signaling pathways such (more ...)