Limits...
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.

Show MeSH

Related in: MedlinePlus

PMA treatment reduced the tyrosine phosphorylation of CBL and dissociation of binding of CRKL to CBL. Immunoprecipitations of U937 cell lysates were performed with anti-CBL antibody after PMA or FcγRI stimulation. Lane 1 & 2 indicates preimmune and resting state (NS) respectively. Lane 3, 4 & 5, treated with PMA (200 ng/ml) for 1, 5 and 10 min. respectively. Lane 6, 7 & 8, stimulated for FcγR1 with 32.2 F(ab)2 and cross linked with secondary antibody (rabbit anti mouse F(ab’)2) for 1 , 5 and 10 min. respectively. Whole cell lysate was added in lane 9 as positive control (WCL). Immunoprecipitated proteins were resolved in 10% SDS-PAGE, blot on nitrocellulose membrane and probed with anti-phosphotyrosine antibody (4G10). This blot was again reprobed with anti-CBL and anti-CRKL antibody. This experiment was repeated three times.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: PMA treatment reduced the tyrosine phosphorylation of CBL and dissociation of binding of CRKL to CBL. Immunoprecipitations of U937 cell lysates were performed with anti-CBL antibody after PMA or FcγRI stimulation. Lane 1 & 2 indicates preimmune and resting state (NS) respectively. Lane 3, 4 & 5, treated with PMA (200 ng/ml) for 1, 5 and 10 min. respectively. Lane 6, 7 & 8, stimulated for FcγR1 with 32.2 F(ab)2 and cross linked with secondary antibody (rabbit anti mouse F(ab’)2) for 1 , 5 and 10 min. respectively. Whole cell lysate was added in lane 9 as positive control (WCL). Immunoprecipitated proteins were resolved in 10% SDS-PAGE, blot on nitrocellulose membrane and probed with anti-phosphotyrosine antibody (4G10). This blot was again reprobed with anti-CBL and anti-CRKL antibody. This experiment was repeated three times.

Mentions: It has been reported by Fernandez et.al.[23] that treatment using PMA decreases tyrosine phosphorylation of CBL in T-cells. In this present study, we have found that PMA stimulation significantly decrease CBL phosphorylation in myeloid cells (Figure 2). As shown in Figure 2, CBL immunoprecipitated from resting state U937IF cells exhibited relatively low level of tyrosine phosphorylation, which was significantly increased upon FcγR1 stimulation. When cells were stimulated with PMA (200 ng/ml), the tyrosine phosphorylation of CBL is markedly decreased after 5 min. (Figure 2, lane 4) and there is no CBL phosphorylation after 10 min. (Figure 2, lane 5). These results establish that the CBL adapter protein underwent a significant decrease in tyrosine phosphorylation after PMA treatment.


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)

PMA treatment reduced the tyrosine phosphorylation of CBL and dissociation of binding of CRKL to CBL. Immunoprecipitations of U937 cell lysates were performed with anti-CBL antibody after PMA or FcγRI stimulation. Lane 1 & 2 indicates preimmune and resting state (NS) respectively. Lane 3, 4 & 5, treated with PMA (200 ng/ml) for 1, 5 and 10 min. respectively. Lane 6, 7 & 8, stimulated for FcγR1 with 32.2 F(ab)2 and cross linked with secondary antibody (rabbit anti mouse F(ab’)2) for 1 , 5 and 10 min. respectively. Whole cell lysate was added in lane 9 as positive control (WCL). Immunoprecipitated proteins were resolved in 10% SDS-PAGE, blot on nitrocellulose membrane and probed with anti-phosphotyrosine antibody (4G10). This blot was again reprobed with anti-CBL and anti-CRKL antibody. This experiment was repeated three times.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: PMA treatment reduced the tyrosine phosphorylation of CBL and dissociation of binding of CRKL to CBL. Immunoprecipitations of U937 cell lysates were performed with anti-CBL antibody after PMA or FcγRI stimulation. Lane 1 & 2 indicates preimmune and resting state (NS) respectively. Lane 3, 4 & 5, treated with PMA (200 ng/ml) for 1, 5 and 10 min. respectively. Lane 6, 7 & 8, stimulated for FcγR1 with 32.2 F(ab)2 and cross linked with secondary antibody (rabbit anti mouse F(ab’)2) for 1 , 5 and 10 min. respectively. Whole cell lysate was added in lane 9 as positive control (WCL). Immunoprecipitated proteins were resolved in 10% SDS-PAGE, blot on nitrocellulose membrane and probed with anti-phosphotyrosine antibody (4G10). This blot was again reprobed with anti-CBL and anti-CRKL antibody. This experiment was repeated three times.
Mentions: It has been reported by Fernandez et.al.[23] that treatment using PMA decreases tyrosine phosphorylation of CBL in T-cells. In this present study, we have found that PMA stimulation significantly decrease CBL phosphorylation in myeloid cells (Figure 2). As shown in Figure 2, CBL immunoprecipitated from resting state U937IF cells exhibited relatively low level of tyrosine phosphorylation, which was significantly increased upon FcγR1 stimulation. When cells were stimulated with PMA (200 ng/ml), the tyrosine phosphorylation of CBL is markedly decreased after 5 min. (Figure 2, lane 4) and there is no CBL phosphorylation after 10 min. (Figure 2, lane 5). These results establish that the CBL adapter protein underwent a significant decrease in tyrosine phosphorylation after PMA treatment.

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