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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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GST-CRKL-SH2 pull down experiment showing the effect of PMA on CBL-CRKL dissociation and CBL dephosphorylation. U937IF cell lysates were prepared and were precipitated with GST fusion protein as described in Methods. 10 μg of GST fusion protein was used for invitro pull down experiments. 50 μl of glutathione-sepharose beads (prewashed with extraction buffer) was added to each sample. GST-Crkl-SH2 fusion (lanes 2–11) protein was used to precipitate associated protein with or without PMA treatment in FcγRI stimulated cell lysate. Lane 1, precipitated with GST alone. Lane 2, no stimulation, Lane 3, 4 & 5, stimulated cell lysate (stimulated with 32.2 F(ab)2 antibody and cross linked with secondary antibody for 1, 5 and 10 min. Lane 6, 7 and 8, cells were treated with PMA (200 ng/ml) for 5 min. followed by FcγR1 stimulations, lane 9, 10 and 11, cells were preincubated with GF109203X (2.5 μM) for 15 min. on ice and then treated with PMA (200 ng/ml) for 5 min. followed by FcγR1 stimulations. Proteins bound to GST fusion protein were resolved in 10% SDS-PAGE, blotted on nitrocellulose membrane and probed with phosphotyrosine antibody (4G10). This blot was reprobed with anti-CBL antibody. This experiment was repeated two times.
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Figure 4: GST-CRKL-SH2 pull down experiment showing the effect of PMA on CBL-CRKL dissociation and CBL dephosphorylation. U937IF cell lysates were prepared and were precipitated with GST fusion protein as described in Methods. 10 μg of GST fusion protein was used for invitro pull down experiments. 50 μl of glutathione-sepharose beads (prewashed with extraction buffer) was added to each sample. GST-Crkl-SH2 fusion (lanes 2–11) protein was used to precipitate associated protein with or without PMA treatment in FcγRI stimulated cell lysate. Lane 1, precipitated with GST alone. Lane 2, no stimulation, Lane 3, 4 & 5, stimulated cell lysate (stimulated with 32.2 F(ab)2 antibody and cross linked with secondary antibody for 1, 5 and 10 min. Lane 6, 7 and 8, cells were treated with PMA (200 ng/ml) for 5 min. followed by FcγR1 stimulations, lane 9, 10 and 11, cells were preincubated with GF109203X (2.5 μM) for 15 min. on ice and then treated with PMA (200 ng/ml) for 5 min. followed by FcγR1 stimulations. Proteins bound to GST fusion protein were resolved in 10% SDS-PAGE, blotted on nitrocellulose membrane and probed with phosphotyrosine antibody (4G10). This blot was reprobed with anti-CBL antibody. This experiment was repeated two times.

Mentions: In order to study CBL-CRKL interaction in U937IF cells following FcγR1 stimulation, we performed pull-down experiments with the GST-CRKL-SH2 domain under conditions of FcγR1 stimulation alone and pretreatment with PMA. Similar to data shown in Figure 2, we observed a basal level of tyrosine phosphorylation of CBL in GST-CRKL-SH2 pull down from resting state (Figure 4, lane 2). Within 1 min. of FcγR1 stimulation a marked increase was seen in amount of tyrosine phosphorylated protein bound to CRKL (Figure 4, compare lane 2 with 3). Pretreatment with PMA followed by FcγR1 cross linking showed decreased amount of phosphorylated CBL pulled down with CRKL-SH2 (Figure 4, lane 6, 7 and 8). To provide further evidence for the role of PKC in tyrosine phosphorylation of CBL through Fcγ receptor desensitization, we performed GST-CRKL-SH2 pull-down experiment with cells pretreated with GF109203X, then stimulated with PMA for 5 min followed by cross linking of FcγR1 at different time periods. Pretreatment with GF109203X partially rescued the PMA induced tyrosine dephosphorylation of CBL (Figure 4 compare lane 9, 10 and 11 with lane 6, 7 and 8). The protective effect of GF109203X on CBL dephosphorylation appeared concomitant to CBL-CRKL association. In addition, we observe a 130 kd tyrosine phosphorylated protein which binds to the CRKL-SH2 domain upon FcγR1 crosslinking and appears to undergo a reduced level of tyrosine phosphorylation upon PMA treatment in U937IF cells (Figure 4). The effect of the PKC inhibitor on tyrosine phosphorylation state of CBL as well as CBL-CRKL interaction suggest that PMA acts through an effect upon tyrosine phosphorylation of CBL following FcγR stimulation.


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)

GST-CRKL-SH2 pull down experiment showing the effect of PMA on CBL-CRKL dissociation and CBL dephosphorylation. U937IF cell lysates were prepared and were precipitated with GST fusion protein as described in Methods. 10 μg of GST fusion protein was used for invitro pull down experiments. 50 μl of glutathione-sepharose beads (prewashed with extraction buffer) was added to each sample. GST-Crkl-SH2 fusion (lanes 2–11) protein was used to precipitate associated protein with or without PMA treatment in FcγRI stimulated cell lysate. Lane 1, precipitated with GST alone. Lane 2, no stimulation, Lane 3, 4 & 5, stimulated cell lysate (stimulated with 32.2 F(ab)2 antibody and cross linked with secondary antibody for 1, 5 and 10 min. Lane 6, 7 and 8, cells were treated with PMA (200 ng/ml) for 5 min. followed by FcγR1 stimulations, lane 9, 10 and 11, cells were preincubated with GF109203X (2.5 μM) for 15 min. on ice and then treated with PMA (200 ng/ml) for 5 min. followed by FcγR1 stimulations. Proteins bound to GST fusion protein were resolved in 10% SDS-PAGE, blotted on nitrocellulose membrane and probed with phosphotyrosine antibody (4G10). This blot was reprobed with anti-CBL antibody. This experiment was repeated two times.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4017086&req=5

Figure 4: GST-CRKL-SH2 pull down experiment showing the effect of PMA on CBL-CRKL dissociation and CBL dephosphorylation. U937IF cell lysates were prepared and were precipitated with GST fusion protein as described in Methods. 10 μg of GST fusion protein was used for invitro pull down experiments. 50 μl of glutathione-sepharose beads (prewashed with extraction buffer) was added to each sample. GST-Crkl-SH2 fusion (lanes 2–11) protein was used to precipitate associated protein with or without PMA treatment in FcγRI stimulated cell lysate. Lane 1, precipitated with GST alone. Lane 2, no stimulation, Lane 3, 4 & 5, stimulated cell lysate (stimulated with 32.2 F(ab)2 antibody and cross linked with secondary antibody for 1, 5 and 10 min. Lane 6, 7 and 8, cells were treated with PMA (200 ng/ml) for 5 min. followed by FcγR1 stimulations, lane 9, 10 and 11, cells were preincubated with GF109203X (2.5 μM) for 15 min. on ice and then treated with PMA (200 ng/ml) for 5 min. followed by FcγR1 stimulations. Proteins bound to GST fusion protein were resolved in 10% SDS-PAGE, blotted on nitrocellulose membrane and probed with phosphotyrosine antibody (4G10). This blot was reprobed with anti-CBL antibody. This experiment was repeated two times.
Mentions: In order to study CBL-CRKL interaction in U937IF cells following FcγR1 stimulation, we performed pull-down experiments with the GST-CRKL-SH2 domain under conditions of FcγR1 stimulation alone and pretreatment with PMA. Similar to data shown in Figure 2, we observed a basal level of tyrosine phosphorylation of CBL in GST-CRKL-SH2 pull down from resting state (Figure 4, lane 2). Within 1 min. of FcγR1 stimulation a marked increase was seen in amount of tyrosine phosphorylated protein bound to CRKL (Figure 4, compare lane 2 with 3). Pretreatment with PMA followed by FcγR1 cross linking showed decreased amount of phosphorylated CBL pulled down with CRKL-SH2 (Figure 4, lane 6, 7 and 8). To provide further evidence for the role of PKC in tyrosine phosphorylation of CBL through Fcγ receptor desensitization, we performed GST-CRKL-SH2 pull-down experiment with cells pretreated with GF109203X, then stimulated with PMA for 5 min followed by cross linking of FcγR1 at different time periods. Pretreatment with GF109203X partially rescued the PMA induced tyrosine dephosphorylation of CBL (Figure 4 compare lane 9, 10 and 11 with lane 6, 7 and 8). The protective effect of GF109203X on CBL dephosphorylation appeared concomitant to CBL-CRKL association. In addition, we observe a 130 kd tyrosine phosphorylated protein which binds to the CRKL-SH2 domain upon FcγR1 crosslinking and appears to undergo a reduced level of tyrosine phosphorylation upon PMA treatment in U937IF cells (Figure 4). The effect of the PKC inhibitor on tyrosine phosphorylation state of CBL as well as CBL-CRKL interaction suggest that PMA acts through an effect upon tyrosine phosphorylation of CBL following FcγR stimulation.

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