Fc receptor activation is associated with rapid tyrosine phosphorylation of CBL adaptor protein, which is complexed via Syk kinase and the γ-subunit of FcγRI[6, 7, 14]. But the interplay between the key signaling molecules which dephosphorylate CBL upon receptor desensitization and hence the deactivation of the phagocytic signalsome are mostly unknown. In the present study, we identified an interaction between PKC and SHP1 which appears to regulate the deactivation of Fcγ receptor leading to the inhibition of the phagocytic signal transduction cascade.
Tyrosine phosphorylation is a critical event for the regulation of signal transduction, cell growth, differentiation and development. Tyrosine phosphorylation of CBL following Fcγ receptor stimulation has been implicated in intracellular signaling pathways via its interaction with several signaling molecules. Tyrosine phosphorylation of CBL has also been demonstrated upon stimulation through T cell receptor[15, 46], B cell antigen receptor[47, 48] granulocyte-macrophage colony stimulating factor and erythropoietin receptor, suggesting that CBL plays an important role in multiple antigen receptor and mitogen receptor associated tyrosine kinase activation pathways in hematopoietic cells.
The increased tyrosine phosphorylation of cellular proteins that occurs after FcγR stimulation is a transient process, which is regulated by the combined action of protein tyrosine kinases and phosphatases. Importantly, once a protein is phosphorylated in vivo on a tyrosine residue, this covalent phosphotyrosine moiety within a given intact protein can only be reversed by the action of a protein tyrosine phosphatase i.e. dephosphorylation (Figure 1). Interestingly, the peak of protein tyrosine phosphorylation in response to Fc receptor activation was observed only after 1 to 5 min. and decreased in next 10–20 min., which suggests that a tyrosine phosphatase was activated following Fcγ receptor engagement. Relatively less work has been done regarding the receptor deactivation mechanisms, including the regulation of phosphatase activity. Our results suggest that tyrosine dephosphorylation of proto-oncoprotein CBL is associated with receptor desensitization and leads to abrogation of the downstream phagocytic signal. These results raise the possibility that a specific tyrosine phosphatase is involved in deactivating the ITAM signaling cascade. Our previous findings and the results observed in the present study indicate that CBL is a substrate for SHP1 and PMA can potentially induce the activation of SHP1. This dephosphorylation of CBL abrogates CBL-CRKL interaction following Fcγ receptor engagement. It has been also suggested that CBL-CRKL interaction is mediated through YXXP motif at the C terminal end of CBL (tyrosine 774). Our mass spectrometry data also confirmed that PMA abrogates CBL-CRKL interaction due to dephosphorylation of CBL at Tyr774 (unpublished observation). Based on these data, we suggest that PMA (probably through SHP1) targets the tyrosine dephosphorylation of CBL at Tyr774 which leads to loss of CBL-CRKL interaction. Several groups have demonstrated that CRKL interacts via its N-terminal SH3 domain with guanine nucleotide exchange factors C3G and SOS[50–52] suggesting that CRKL activates small G-protein signaling pathways. In Jurkat T cells, CBL through its interaction with CRKL protein becomes coupled to a guanine nucleotide exchange factor. These findings strongly suggest the possibility that tyrosine phosphorylation of CBL may provide one mechanism to link upstream tyrosine kinase to small G protein regulation. These finding are consistent with the result that PMA abrogates ITAM induced Rac-GTP activity (Figure 7), a biochemical phenomenon that has been implicated in the regulation of cytoskeleton rearrangement and phagocytosis.
PMA is known to activate PKCs by binding to their cysteine rich domain and facilitating their translocation to plasma membrane[53–55]. Using PKC specific inhibitor GF109203X (inhibits both classical and novel PKCs) the inhibitory effect of PMA on phosphorylation of CBL and phagocytosis was reversed, indicating the involvement of PKC in this phenomenon. The inhibition of PKC activity by GF109203X, leads to higher tyrosine phosphorylation of other cellular proteins. It has also been reported by others that PMA induces dephosphorylation of Shc in T cells and this can be reversed by GF109203X indicating that PKC activation modulates the tyrosine phosphorylation state of other cellular protein besides CBL. It has also been reported previously by others that PMA causes serine/threonine phosphorylation of growth factor receptor and serine phosphorylation of IRS1 resulting an inhibition of receptor protein tyrosine kinase activity[56, 57]. Liu et al. have reported that PMA induces the serine phosphorylation of CBL and promote the association of Tau isoform of 14-3-3 and this serine phosphorylation of CBL suppressed its tyrosine phosphorylation by tyrosine kinase inhibition, however the mechanism is not yet known. It has also been reported that PKC does not bind to CBL directly, raising the possibility that PKC might indirectly regulate tyrosine phosphorylation of CBL by bringing a tyrosine phosphatase into the signalsome, by activating a specific PTP or by altering substrate availability to kinases and/or phosphatases. Our results provide evidence that PKC controls the tyrosine phosphorylation state of CBL via a mechanism that somehow requires the protein tyrosine phosphatase activity of SHP1.
In the present study, we examined the role of PKC in the control of Fcγ receptor mediated phagocytosis. The data presented here demonstrate that Fcγ receptor mediated phagocytosis was markedly inhibited by exposure of cells to PMA, which was associated with CBL tyrosine dephosphorylation and downstream inhibition of Rac-GTP activation. The literature contains conflicting reports concerning the role of PKC in phagocytosis. Pharmacological inhibition or expression of dominant negative isoforms of PKC reduced phagocytosis in several systems[59–61]. However, the precise role of the particular PKC isoforms involved in phagocytosis remains unclear. Involvement of PKC activity in complement receptor-mediated phagocytosis has been clearly demonstrated. In the case of FcγR-mediated phagocytosis, data are more complex. Differences in these reports regarding the involvement of PKC may be a result of the differential role of various PKC isoforms in phagocytosis. In U937 monocytes, it was found that FcγRI engagement leads to an increase in PKC activity that is Ca2+-independent and corresponds to translocation to the membrane of the PKC isoforms δ, ϵ, and ζ. In U937-differentiated macrophages, FcγRI engagement leads to PKC activity that is Ca2+-dependent and corresponds to membrane translocation of the conventional PKC isoforms α, β, and γ[64, 65].
Zheleznyak and Brown have found that activation of PKC is an early signal required for Fc receptor mediated phagocytosis in human monocytes. It is possible that PKC may play a different role in Fcγ receptor signaling and phagocytosis at different time points following receptor engagement as is true for many signaling molecules. We and others have demonstrated that tyrosine phosphorylation is a critical signaling event that underlies Fcγ receptor mediated phagocytosis in mouse macrophages and the formation of tyrosine phosphorylation coincides with the appearance of F-actin beneath the phagocytic cup.[29, 62, 67]. They reported that genestein (tyrosine kinase inhibitor) but not inhibitors of protein kinase C, block the ingestion process during phagocytosis. In agreement with these data, we observed that PMA induces Fc receptor mediated rapid CBL dephosphorylation which coincides with abrogation of phagocytosis. It has also been reported by Romanova et al. that Rac1 is necessary for the IL3 induced assembly of membrane ruffles in Baf3 (human pre-B lymphoid) cell line and PMA dissolves the actin formed membrane ruffles and round the cells in presence of IL-3. Rac is directly involved in actin polymerization/formation of lamellipodia. It plays an important role in engulfment in phagocytosis. Our results suggest that PMA induced a dephosphorylation of CBL which is associated with a block in the downstream signaling cascade, the conversion of Rac-GDP to Rac-GTP, an event that is essential for the phagocytic response.
Tyrosine phosphorylation is controlled by the coordinated action of PTKs (protein tyrosine kinase) and protein tyrosine phosphates (PTPs)[69–71]. The work from our laboratory has reported that SHP1 inhibits Fcγ receptors mediated phagocytosis by altering the phosphorylation state of CBL and blocked Rac-GTP. In macrophages, SHP1 also selectively regulates the tyrosine phosphorylation of Stat1 and Jak1 while leaving Tyk2 and Stat2 unaffected.
Herein, we present evidence that PKC negatively regulates the tyrosine phosphorylation state of the CBL adapter protein and inhibits FcγR dependent ITAM signaling and phagocytosis. Our experiments performed with heterologous expression of SHP1 vs. the phosphatase-dead SHP1 support the notion that this PKC mediated event is dependent upon SHP1 phosphatase activity. Previous reports from our laboratory and others have confirmed a role of CBL in the regulation of FcγR mediated phagocytosis and that SHP1 associates with CBL.