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A PKC-SHP1 signaling axis desensitizes Fcγ receptor signaling by reducing the tyrosine phosphorylation of CBL and regulates FcγR mediated phagocytosis.

Joshi S, Singh AR, Zulcic M, Durden DL - BMC Immunol. (2014)

Bottom Line: Pretreatment of J774 cells with GF109203X, a PKC inhibitor was observed to block dephosphorylation of CBL and rescued the phagocytic response.We demonstrate that the PKC induced desensitization of FcγR/ phagocytosis is associated with the inactivation of Rac-GTP, which is deactivated in a hematopoietic specific phosphatase SHP1 dependent manner following ITAM stimulation.The effect of PKC on FcγR signaling is augmented by the transfection of catalytically active SHP1 and not by the transfection of catalytic dead SHP1 (C124S).

View Article: PubMed Central - HTML - PubMed

Affiliation: UCSD Department of Pediatrics, Moores UCSD Cancer Center, University of California School of Medicine, San Diego, CA 92093, USA. ddurden@ucsd.edu.

ABSTRACT

Background: Fcγ receptors mediate important biological signals in myeloid cells including the ingestion of microorganisms through a process of phagocytosis. It is well-known that Fcγ receptor (FcγR) crosslinking induces the tyrosine phosphorylation of CBL which is associated with FcγR mediated phagocytosis, however how signaling molecules coordinate to desensitize these receptors is unclear. An investigation of the mechanisms involved in receptor desensitization will provide new insight into potential mechanisms by which signaling molecules may downregulate tyrosine phosphorylation dependent signaling events to terminate important signaling processes.

Results: Using the U937IF cell line, we observed that FcγR1 crosslinking induces the tyrosine phosphorylation of CBL, which is maximal at 5 min. followed by a kinetic pattern of dephosphorylation. An investigation of the mechanisms involved in receptor desensitization revealed that pretreatment of U937IF or J774 cells with PMA followed by Fcγ receptor crosslinking results in the reduced tyrosine phosphorylation of CBL and the abrogation of downstream signals, such as CBL-CRKL binding, Rac-GTP activation and the phagocytic response. Pretreatment of J774 cells with GF109203X, a PKC inhibitor was observed to block dephosphorylation of CBL and rescued the phagocytic response. We demonstrate that the PKC induced desensitization of FcγR/ phagocytosis is associated with the inactivation of Rac-GTP, which is deactivated in a hematopoietic specific phosphatase SHP1 dependent manner following ITAM stimulation. The effect of PKC on FcγR signaling is augmented by the transfection of catalytically active SHP1 and not by the transfection of catalytic dead SHP1 (C124S).

Conclusions: Our results suggest a functional model by which PKC interacts with SHP1 to affect the phosphorylation state of CBL, the activation state of Rac and the negative regulation of ITAM signaling i.e. Fcγ receptor mediated phagocytosis. These findings suggest a mechanism for Fcγ receptor desensitization by which a serine-threonine kinase e.g. PKC downregulates tyrosine phosphorylation dependent signaling events via the reduced tyrosine phosphorylation of the complex adapter protein, CBL.

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Fcγ Receptor induced phagocytic activity is decreased following the treatment by PMA in J774A.1 cell line. (A) For PMA treatment, cells were pre incubated with PMA (0.2 μg/ml or 0.4 μg/ml) for 15 min. before the onset of phagocytosis. In other set, cells were pre incubated with GF109203X for 15 min. followed by PMA (0.4 μg/ml) treatment. To initiate phagocytosis, media was replaced by 1ml IgG coated sheep RBCs and incubated for 30 min. at 37oC. (B) Effect of over expression of SHP-1 on CBL dephosphorylation.. J774A.1 cells were infected either with empty vector recombinant virus (pSC 65) or wild type SHP-1 (MOI 2 pfu/cell for overnight) or with C2 mutant of SHP-1. P and NS represents pre-immune and non-stimulation respectively, non infected serve as control. Except P and NS conditions, all cells were stimulated with sensitized sheep RBCs for 10 min. Immunoblots were reprobed with anti-CBL, anti-SHP-1 and anti-CRKL antibody. Lower panel shows the Western blot analysis of over expression of SHP-1 and C2 mutant in J774A.1 cells. (C) J774A-1 cells were infected with pSC 65or recombinant vaccinia for the expression of C2 mutant or SHP-1 at an MOI of 2 pfu / cell for 4 hours at 37oC as described in Methods. Western blot analysis confirms equivalent level of over expression of SHP-1 and C2 mutant in J774-1 cells. (D). Peritoneal macrophages isolated from C57BL/6 mice were pre incubated with PMA for 15 min. before the onset of phagocytosis. In other set, cells were pre incubated with GF109203X (above mentioned conc.) for 15 min. followed by PMA (0.4 μg/ml) treatment and phagocytosis was performed as described above. Each bar represents mean ± SD, n=3. *P <0.05, **P <0.01 and ***P <0.001 vs. control (t-test) All these experiments were performed three times.
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Figure 6: Fcγ Receptor induced phagocytic activity is decreased following the treatment by PMA in J774A.1 cell line. (A) For PMA treatment, cells were pre incubated with PMA (0.2 μg/ml or 0.4 μg/ml) for 15 min. before the onset of phagocytosis. In other set, cells were pre incubated with GF109203X for 15 min. followed by PMA (0.4 μg/ml) treatment. To initiate phagocytosis, media was replaced by 1ml IgG coated sheep RBCs and incubated for 30 min. at 37oC. (B) Effect of over expression of SHP-1 on CBL dephosphorylation.. J774A.1 cells were infected either with empty vector recombinant virus (pSC 65) or wild type SHP-1 (MOI 2 pfu/cell for overnight) or with C2 mutant of SHP-1. P and NS represents pre-immune and non-stimulation respectively, non infected serve as control. Except P and NS conditions, all cells were stimulated with sensitized sheep RBCs for 10 min. Immunoblots were reprobed with anti-CBL, anti-SHP-1 and anti-CRKL antibody. Lower panel shows the Western blot analysis of over expression of SHP-1 and C2 mutant in J774A.1 cells. (C) J774A-1 cells were infected with pSC 65or recombinant vaccinia for the expression of C2 mutant or SHP-1 at an MOI of 2 pfu / cell for 4 hours at 37oC as described in Methods. Western blot analysis confirms equivalent level of over expression of SHP-1 and C2 mutant in J774-1 cells. (D). Peritoneal macrophages isolated from C57BL/6 mice were pre incubated with PMA for 15 min. before the onset of phagocytosis. In other set, cells were pre incubated with GF109203X (above mentioned conc.) for 15 min. followed by PMA (0.4 μg/ml) treatment and phagocytosis was performed as described above. Each bar represents mean ± SD, n=3. *P <0.05, **P <0.01 and ***P <0.001 vs. control (t-test) All these experiments were performed three times.

Mentions: Since several studies have recently reported the requirement for specific tyrosine kinases, Syk and Src family kinases in phagocytosis mediated events[32,33,36,45], we presumed that dephosphorylation of specific cell protein on specific tyrosine phosphorylation sites may negatively regulate phagocytosis. We previously reported that Fcγ receptor cross-linking induces tyrosine phosphorylation of the complex adapter protein CBL[11]. More recent data from our laboratory provide evidence that CBL is required in FcγR mediated phagocytic response[24]. These observations prompted us to study the role of tyrosine phosphorylation of CBL in the induction of phagocytic signaling. To address this question, we have studied the tyrosine phosphorylation of CBL in J774 A.1 cells stimulated with IgG sensitized sheep RBC. Figure 5 shows that tyrosine phosphorylation of CBL is attenuated in PMA pre-treated cells upon Fcγ receptor stimulation (lane 6, 7 and 8). Phagocytosis of IgG coated sRBC (Figure 6A) was found significantly inhibited following treatment of PMA in J774A.1 cells. This inhibitory effect of PMA was blocked by the PKC inhibitor, GF109203X (Figure 6A). These data suggest that FcγR induced phagocytic event is regulated by PKC. Previous work in our laboratory demonstrated that SHP-1 overexpression inhibits FcγR mediated phagocytosis compared to no effect of a catalytically dead mutant of SHP-1 and heterologous overexpression of wild type SHP-1 in J774 A.1 enhances the dephosphorylation of CBL[24] . These results combined with our observation that PMA induced the dephosphorylation of CBL prompted us to examine the crosstalk between PKC and SHP-1 on FcγR mediated downstream event. To execute these experiments, J774A.1 cells were infected either with empty vector recombinant vaccinia virus (pSC65) or recombinant vaccinia virus for the expression of wild type SHP-1 or catalytically dead SHP-1 at an MOI 2 pfu/cell for 4 hours at 37°C. The result shows that effect of SHP-1 overexpression on phagocytosis is blocked by the inhibition of PKC (treatment with GF109203X) (Figure 6C). Importantly, PMA has no effect on Fcγ receptor mediated phagocytosis when cells were overexpressing the catalytically dead SHP1 (C124S). Hence, we suggest that PKC desensitizes Fcγ receptor in a SHP1 tyrosine phosphatase dependent manner resulting in the dephosphorylation of CBL an effect which blocks the downstream phagocytosis response.


A PKC-SHP1 signaling axis desensitizes Fcγ receptor signaling by reducing the tyrosine phosphorylation of CBL and regulates FcγR mediated phagocytosis.

Joshi S, Singh AR, Zulcic M, Durden DL - BMC Immunol. (2014)

Fcγ Receptor induced phagocytic activity is decreased following the treatment by PMA in J774A.1 cell line. (A) For PMA treatment, cells were pre incubated with PMA (0.2 μg/ml or 0.4 μg/ml) for 15 min. before the onset of phagocytosis. In other set, cells were pre incubated with GF109203X for 15 min. followed by PMA (0.4 μg/ml) treatment. To initiate phagocytosis, media was replaced by 1ml IgG coated sheep RBCs and incubated for 30 min. at 37oC. (B) Effect of over expression of SHP-1 on CBL dephosphorylation.. J774A.1 cells were infected either with empty vector recombinant virus (pSC 65) or wild type SHP-1 (MOI 2 pfu/cell for overnight) or with C2 mutant of SHP-1. P and NS represents pre-immune and non-stimulation respectively, non infected serve as control. Except P and NS conditions, all cells were stimulated with sensitized sheep RBCs for 10 min. Immunoblots were reprobed with anti-CBL, anti-SHP-1 and anti-CRKL antibody. Lower panel shows the Western blot analysis of over expression of SHP-1 and C2 mutant in J774A.1 cells. (C) J774A-1 cells were infected with pSC 65or recombinant vaccinia for the expression of C2 mutant or SHP-1 at an MOI of 2 pfu / cell for 4 hours at 37oC as described in Methods. Western blot analysis confirms equivalent level of over expression of SHP-1 and C2 mutant in J774-1 cells. (D). Peritoneal macrophages isolated from C57BL/6 mice were pre incubated with PMA for 15 min. before the onset of phagocytosis. In other set, cells were pre incubated with GF109203X (above mentioned conc.) for 15 min. followed by PMA (0.4 μg/ml) treatment and phagocytosis was performed as described above. Each bar represents mean ± SD, n=3. *P <0.05, **P <0.01 and ***P <0.001 vs. control (t-test) All these experiments were performed three times.
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Figure 6: Fcγ Receptor induced phagocytic activity is decreased following the treatment by PMA in J774A.1 cell line. (A) For PMA treatment, cells were pre incubated with PMA (0.2 μg/ml or 0.4 μg/ml) for 15 min. before the onset of phagocytosis. In other set, cells were pre incubated with GF109203X for 15 min. followed by PMA (0.4 μg/ml) treatment. To initiate phagocytosis, media was replaced by 1ml IgG coated sheep RBCs and incubated for 30 min. at 37oC. (B) Effect of over expression of SHP-1 on CBL dephosphorylation.. J774A.1 cells were infected either with empty vector recombinant virus (pSC 65) or wild type SHP-1 (MOI 2 pfu/cell for overnight) or with C2 mutant of SHP-1. P and NS represents pre-immune and non-stimulation respectively, non infected serve as control. Except P and NS conditions, all cells were stimulated with sensitized sheep RBCs for 10 min. Immunoblots were reprobed with anti-CBL, anti-SHP-1 and anti-CRKL antibody. Lower panel shows the Western blot analysis of over expression of SHP-1 and C2 mutant in J774A.1 cells. (C) J774A-1 cells were infected with pSC 65or recombinant vaccinia for the expression of C2 mutant or SHP-1 at an MOI of 2 pfu / cell for 4 hours at 37oC as described in Methods. Western blot analysis confirms equivalent level of over expression of SHP-1 and C2 mutant in J774-1 cells. (D). Peritoneal macrophages isolated from C57BL/6 mice were pre incubated with PMA for 15 min. before the onset of phagocytosis. In other set, cells were pre incubated with GF109203X (above mentioned conc.) for 15 min. followed by PMA (0.4 μg/ml) treatment and phagocytosis was performed as described above. Each bar represents mean ± SD, n=3. *P <0.05, **P <0.01 and ***P <0.001 vs. control (t-test) All these experiments were performed three times.
Mentions: Since several studies have recently reported the requirement for specific tyrosine kinases, Syk and Src family kinases in phagocytosis mediated events[32,33,36,45], we presumed that dephosphorylation of specific cell protein on specific tyrosine phosphorylation sites may negatively regulate phagocytosis. We previously reported that Fcγ receptor cross-linking induces tyrosine phosphorylation of the complex adapter protein CBL[11]. More recent data from our laboratory provide evidence that CBL is required in FcγR mediated phagocytic response[24]. These observations prompted us to study the role of tyrosine phosphorylation of CBL in the induction of phagocytic signaling. To address this question, we have studied the tyrosine phosphorylation of CBL in J774 A.1 cells stimulated with IgG sensitized sheep RBC. Figure 5 shows that tyrosine phosphorylation of CBL is attenuated in PMA pre-treated cells upon Fcγ receptor stimulation (lane 6, 7 and 8). Phagocytosis of IgG coated sRBC (Figure 6A) was found significantly inhibited following treatment of PMA in J774A.1 cells. This inhibitory effect of PMA was blocked by the PKC inhibitor, GF109203X (Figure 6A). These data suggest that FcγR induced phagocytic event is regulated by PKC. Previous work in our laboratory demonstrated that SHP-1 overexpression inhibits FcγR mediated phagocytosis compared to no effect of a catalytically dead mutant of SHP-1 and heterologous overexpression of wild type SHP-1 in J774 A.1 enhances the dephosphorylation of CBL[24] . These results combined with our observation that PMA induced the dephosphorylation of CBL prompted us to examine the crosstalk between PKC and SHP-1 on FcγR mediated downstream event. To execute these experiments, J774A.1 cells were infected either with empty vector recombinant vaccinia virus (pSC65) or recombinant vaccinia virus for the expression of wild type SHP-1 or catalytically dead SHP-1 at an MOI 2 pfu/cell for 4 hours at 37°C. The result shows that effect of SHP-1 overexpression on phagocytosis is blocked by the inhibition of PKC (treatment with GF109203X) (Figure 6C). Importantly, PMA has no effect on Fcγ receptor mediated phagocytosis when cells were overexpressing the catalytically dead SHP1 (C124S). Hence, we suggest that PKC desensitizes Fcγ receptor in a SHP1 tyrosine phosphatase dependent manner resulting in the dephosphorylation of CBL an effect which blocks the downstream phagocytosis response.

Bottom Line: Pretreatment of J774 cells with GF109203X, a PKC inhibitor was observed to block dephosphorylation of CBL and rescued the phagocytic response.We demonstrate that the PKC induced desensitization of FcγR/ phagocytosis is associated with the inactivation of Rac-GTP, which is deactivated in a hematopoietic specific phosphatase SHP1 dependent manner following ITAM stimulation.The effect of PKC on FcγR signaling is augmented by the transfection of catalytically active SHP1 and not by the transfection of catalytic dead SHP1 (C124S).

View Article: PubMed Central - HTML - PubMed

Affiliation: UCSD Department of Pediatrics, Moores UCSD Cancer Center, University of California School of Medicine, San Diego, CA 92093, USA. ddurden@ucsd.edu.

ABSTRACT

Background: Fcγ receptors mediate important biological signals in myeloid cells including the ingestion of microorganisms through a process of phagocytosis. It is well-known that Fcγ receptor (FcγR) crosslinking induces the tyrosine phosphorylation of CBL which is associated with FcγR mediated phagocytosis, however how signaling molecules coordinate to desensitize these receptors is unclear. An investigation of the mechanisms involved in receptor desensitization will provide new insight into potential mechanisms by which signaling molecules may downregulate tyrosine phosphorylation dependent signaling events to terminate important signaling processes.

Results: Using the U937IF cell line, we observed that FcγR1 crosslinking induces the tyrosine phosphorylation of CBL, which is maximal at 5 min. followed by a kinetic pattern of dephosphorylation. An investigation of the mechanisms involved in receptor desensitization revealed that pretreatment of U937IF or J774 cells with PMA followed by Fcγ receptor crosslinking results in the reduced tyrosine phosphorylation of CBL and the abrogation of downstream signals, such as CBL-CRKL binding, Rac-GTP activation and the phagocytic response. Pretreatment of J774 cells with GF109203X, a PKC inhibitor was observed to block dephosphorylation of CBL and rescued the phagocytic response. We demonstrate that the PKC induced desensitization of FcγR/ phagocytosis is associated with the inactivation of Rac-GTP, which is deactivated in a hematopoietic specific phosphatase SHP1 dependent manner following ITAM stimulation. The effect of PKC on FcγR signaling is augmented by the transfection of catalytically active SHP1 and not by the transfection of catalytic dead SHP1 (C124S).

Conclusions: Our results suggest a functional model by which PKC interacts with SHP1 to affect the phosphorylation state of CBL, the activation state of Rac and the negative regulation of ITAM signaling i.e. Fcγ receptor mediated phagocytosis. These findings suggest a mechanism for Fcγ receptor desensitization by which a serine-threonine kinase e.g. PKC downregulates tyrosine phosphorylation dependent signaling events via the reduced tyrosine phosphorylation of the complex adapter protein, CBL.

Show MeSH
Related in: MedlinePlus