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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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Specific PKC inhibitor (GF109203X) maintains the basal tyrosine phosphorylation state of CBL. Human IFN γ differentiated U937 cells were treated with or without GF 109203X (2.5 μM) on ice for 15 min. and then stimulated with PMA (200 ng/ml) for 1, 5 & 10 min. at 37°C. Cell lysates were prepared and immunoprecipitated (IP) with anti-CBL antibody. Lane 1 represents precipitation with preimmune antisera. Lane 2, no stimulation. Lane 3, 4 & 5 and 9, 10 & 11 represent U937 cells stimulated with PMA (200 ng/ml) or 32.2 F(ab)2 for 1, 5 & 10 min. respectively. Lane 6, 7 & 8, represents cells preincubated with GF 109203X followed by PMA stimulation. This blot was reprobed with anti-CBL and anti-CRKL antibody.
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Figure 3: Specific PKC inhibitor (GF109203X) maintains the basal tyrosine phosphorylation state of CBL. Human IFN γ differentiated U937 cells were treated with or without GF 109203X (2.5 μM) on ice for 15 min. and then stimulated with PMA (200 ng/ml) for 1, 5 & 10 min. at 37°C. Cell lysates were prepared and immunoprecipitated (IP) with anti-CBL antibody. Lane 1 represents precipitation with preimmune antisera. Lane 2, no stimulation. Lane 3, 4 & 5 and 9, 10 & 11 represent U937 cells stimulated with PMA (200 ng/ml) or 32.2 F(ab)2 for 1, 5 & 10 min. respectively. Lane 6, 7 & 8, represents cells preincubated with GF 109203X followed by PMA stimulation. This blot was reprobed with anti-CBL and anti-CRKL antibody.

Mentions: To determine the specific effect of PKC on tyrosine phosphorylation of CBL, U937IF cells were pre-incubated with a specific PKC inhibitor, GF109203X, followed by PMA treatment. Our data suggest that CBL phosphorylation was maintained for 1 to 5 min. (Figure 3, lane 6 and 7) in GF109203X treated cells pre-incubated with PMA in comparison to cells treated with PMA alone (Figure 3, lanes 3, 4 and 5). In addition to CBL phosphorylation, CBL-CRKL association was also maintained in cells pre-incubated with GF109203X.


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)

Specific PKC inhibitor (GF109203X) maintains the basal tyrosine phosphorylation state of CBL. Human IFN γ differentiated U937 cells were treated with or without GF 109203X (2.5 μM) on ice for 15 min. and then stimulated with PMA (200 ng/ml) for 1, 5 & 10 min. at 37°C. Cell lysates were prepared and immunoprecipitated (IP) with anti-CBL antibody. Lane 1 represents precipitation with preimmune antisera. Lane 2, no stimulation. Lane 3, 4 & 5 and 9, 10 & 11 represent U937 cells stimulated with PMA (200 ng/ml) or 32.2 F(ab)2 for 1, 5 & 10 min. respectively. Lane 6, 7 & 8, represents cells preincubated with GF 109203X followed by PMA stimulation. This blot was reprobed with anti-CBL and anti-CRKL antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4017086&req=5

Figure 3: Specific PKC inhibitor (GF109203X) maintains the basal tyrosine phosphorylation state of CBL. Human IFN γ differentiated U937 cells were treated with or without GF 109203X (2.5 μM) on ice for 15 min. and then stimulated with PMA (200 ng/ml) for 1, 5 & 10 min. at 37°C. Cell lysates were prepared and immunoprecipitated (IP) with anti-CBL antibody. Lane 1 represents precipitation with preimmune antisera. Lane 2, no stimulation. Lane 3, 4 & 5 and 9, 10 & 11 represent U937 cells stimulated with PMA (200 ng/ml) or 32.2 F(ab)2 for 1, 5 & 10 min. respectively. Lane 6, 7 & 8, represents cells preincubated with GF 109203X followed by PMA stimulation. This blot was reprobed with anti-CBL and anti-CRKL antibody.
Mentions: To determine the specific effect of PKC on tyrosine phosphorylation of CBL, U937IF cells were pre-incubated with a specific PKC inhibitor, GF109203X, followed by PMA treatment. Our data suggest that CBL phosphorylation was maintained for 1 to 5 min. (Figure 3, lane 6 and 7) in GF109203X treated cells pre-incubated with PMA in comparison to cells treated with PMA alone (Figure 3, lanes 3, 4 and 5). In addition to CBL phosphorylation, CBL-CRKL association was also maintained in cells pre-incubated with GF109203X.

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