PYK2 interacts with MyD88 and regulates MyD88-mediated NF-kappaB activation in macrophages

Abstract

PYK2, a major cell adhesion-activated tyrosine kinase, is highly expressed in macrophages and implicated in macrophage activation and inflammatory response. However, mechanisms by which PYK2 regulates inflammatory response are beginning to be understood. In this study, we demonstrate that PYK2 interacts with MyD88, a crucial signaling adaptor protein in LPS and PGN-induced NF-kappaB activation, in vitro and in macrophages. This interaction, increased in macrophages, stimulated by LPS, requires the death domain of MyD88. PYK2-deficient macrophages exhibit reduced phosphorylation and degradation of IkappaB, an inhibitor of NF-kappaB nuclear translocation, and decreased NF-kappaB activation and IL-1beta expression by LPS. These results suggest that via interaction with MyD88, PYK2 is involved in modulating cytokine (e.g., LPS) stimulation of NF-kappaB activity and signaling, providing a mechanism underlying PYK2 regulation of an inflammatory response.

Figure 1.

PYK2 interaction with MyD88. (A) PYK2 interaction with MyD88 in vitro via PYK2 C-terminal domain. Cos-7 cells expressing Flag-tagged MyD88 were lysed, and resulting lysates were incubated with indicated GST fusion proteins (∼5 μg) immobilized on beads. Bound proteins were probed with anti-Flag antibodies (upper right). GST fusion proteins were revealed by Coomassie staining (lower right). The data were summarized (left). ++, Strong binding; +, weak binding; –, no binding. FAT, Focal adhesion targeting domain; IB, immunoblot. (B) Coimmunoprecipitation (IP) of MyD88 with PYK2 that is regulated by PYK2 catalytic activity. (C) A working model to illustrate enhanced PYK2-MyD88 interaction by PYK2 catalytic activity. FERM, Protein 4.1, ezrin, radixin, moesin. (D) Weak tyrosine phosphorylation of MyD88 in cells coexpressing PYK2 but not FAK. (B and D) HEK-293 cells were transiently transfected with indicated plasmids. Cell lysates were incubated with anti-Flag antibodies to immunoprecipitate MyD88 complexes, which were resolved on SDS-PAGE and immunoblotted with antibodies against Myc or RC20 [a phosphotyrosine antibody (Ptyr)]. Flag-tagged MyD88 and Myc-tagged FAK/PYK2 in lysates were revealed by immunoblotting with anti-Flag and anti-Myc antibodies, respectively.

Figure 2.

Mapping PYK2 interaction domain in MyD88. (A) Illustration of MyD88 deletion mutants and its interaction with PYK2. ++, Strong interaction/inhibition of PY402; –, no detectable interaction/no decrease of PY402. (B) Coimmunoprecipitation of PYK2 with MyD88 deletion mutants. HEK-293 cells were transiently transfected with indicated plasmids. Cell lysates were incubated with anti-Flag antibodies to immunoprecipitate MyD88 complexes, which were resolved on SDS-PAGE and immunoblotted with antibodies against Myc. Flag-tagged MyD88 and Myc-tagged PYK2 in lysates were revealed by immunoblotting with anti-Flag and anti-Myc antibodies, respectively. In addition, PY402 was also examined. Note that deletion of the DD abolished the interaction. Interestingly, PY402 was decreased in cells coexpressing MyD88, but not the MyD88ΔDD mutant. These results were summarized in A.

Figure 3.

PYK2 interaction with MyD88 in macrophages. (A and B) Coimmunoprecipitation of MyD88 with PYK2 in RAW264.7 cell lysates stimulated with or without LPS (1 μg/ml, 10 min). Lysates (5 μg) were loaded as input. IgG-NS, IgG-nonspecific. Original bar, 20 μm. (C) Coimmunostaining analysis of PYK2 and MyD88 in RAW264.7 cells, which were fixed and immunostained with anti-PYK2 and anti-MyD88 antibodies. (D) Western blot analysis of LPS-induced PYK2 tyrosine phosphorylation indicated antibodies.

Figure 4.

Requirement of PYK2 for LPS- and PGN-induced phosphorylation and degradation of IκB. (A) Western blot analysis of RAW264.7 macrophages stably expressing control shRNA and PYK2 shRNA using indicated antibodies. Note that PYK2 is selectively reduced in shRNA-PYK2 but not in control shRNA-expressing cells. FAK, paxillin, and p130^cas were not affected in PYK2-shRNA-expressing cells. (B) Impaired time course of LPS-induced phosphorylation (p) and degradation of IκB in PYK2-deficient RAW264.7 cells, which when expressing control and shRNA-PYK2, were stimulated with LPS (1 μg/ml) for the indicated time. Cell lysates were subjected for Western blot analysis using indicated antibodies. Levels of total β-actin were used as controls for protein loading. (C) Quantification analysis of data from B. The values displayed are combined from at least three separate experiments and are expressed as fold increase over the basal values without stimulation. *, ^#, P < 0.01, significant decrease/increase from the control (t-test). (D) Reduced PGN induced phosphorylation of IκB in PYK2-deficient RAW264.7 cells, which when expressing control and PYK2 shRNA, were stimulated with or without PGN (10 ng/ml) for different times. Lysates were subjected to Western blot analysis using indicated antibodies. (E) Quantification analysis of data from D. The values displayed are combined from at least two separate experiments and are expressed as fold increase over the basal values without stimulation. *, P < 0.01, significant difference from the control (t-test).

Figure 5.

Reduced LPS-induced p65 nuclear translocation in RAW264.7 cells expressing miRNA-PYK2. (A) Western blot analysis showing PYK2 expression in Cos-7 cells cotransfected with indicated plasmids. Note that miRNA-PYK2-2 specifically suppresses exogenous PYK2 expression. (B) Immunostaining analysis of endogenous PYK2 in RAW264.7 cells transfected with the scramble and miRNA-PYK2-2. RAW264.7 cells expressing the miRNA plasmid, which encodes GFP as an indicator, were fixed and immunostained with anti-PYK2 antibody (monoclonal; red). Open arrows indicate control GFP expression cells, and filled arrows indicate reduced PYK2 expression in miRNA-PYK2-transfected cells. (C) LPS (1 μg/ml)-induced p65 nuclear translocation in RAW264.7 cells expressing with (filled arrows) or without (open arrows) miRNA-PYK2 were examined by immunostaining analysis using indicated antibodies. Original marker bars, 20 μm. DAPI, 4′,6-Diamidino-2-phenylindole. (D) Quantification analysis of data from C. The ratio of p65 immunofluorescence intensity in nuclei over cytoplasm was presented as means ± sd (n=10). *, P < 0.01, in comparison with cells without miRNA-PYK2 expression.

Figure 6.

Impaired LPS- and PGN-induced p65 nuclear translocation in PYK2-deficient RAW264.7 cell lines. (A) Immunofluorescene analysis of p65 distribution in the control and PYK2-shRNA cell lines stimulated without (Control) or with LPS (1 μg/ml, 20 min) or PGN (10 ng/ml, 20 min). Original marker bar, 20 μm. Open arrows indicate p65 staining at the cytoplasm, and filled arrows indicate p65 distribution at the nuclei. (B) Quantification analysis of data from A. The ratio of p65 immunofluorescence intensity in nuclei over cytoplasm was presented as means ± sd (n=10). *, P < 0.01, in comparison with the control cells without LPS/PGN stimulation.

Figure 7.

PYK2 regulation of NF-κB promoter activity and NF-κB-dependent IL-1β expression. (A) PYK2 is sufficient to activate NF-κB promoter. (B) PYK2 is necessary for LPS-induced, NF-κB-dependent IL-1β expression. (A) RAW264.7 cells were transiently transfected with PYK2 or PYK2 mutants with NF-κB-luciferase and β-galactosidase. (B) Control and PYK2-shRNA-expressing RAW264.7 cells were stimulated with or without LPS (1 μg/ml, 1 h). The mRNAs from treated cells were analyzed by real-time PCR for gene expression of IL-1β. The values displayed are combined from at least three separate experiments and are expressed as fold increase over the control (β-actin). PYK2, but not PYK2 mutants, increases luciferase production, illustrated in A. LPS induces IL-1β production in the control, but not PYK2-shRNA cells, illustrated in B. *, P < 0.01, in comparison with the control vector (A) or control without LPS stimulation (B).

Figure 8.

A working hypothesis for PYK2 regulation of LPS/PGN-induced NF-κB activation by its interaction with MyD88.