M-CSF Induces Phosphorylation of NF-κB Ser276 in a PKC-dependent Fashion
Since M-CSF did not regulate NF-κB activation by influencing IκBα, we next sought to determine if M-CSF affected NF-κB p65 by post-translational mechanisms. Thus, we examined the phosphorylation of NF-κB p65 with specific phospho-NFκB p65 (Ser276 and Ser536) antibodies. M-CSF induced the phosphorylation of Ser276 but not Ser536 of NF-κB p65 in MDMs. Compared to vehicle, the general PKC inhibitor Ro-31-8220 reduced Ser276 phosphorylation, but not Ser536, phosphorylation in M-CSF-stimulated cells (Figure 5B). Furthermore, M-CSF-stimulated NF-κB p65 phosphorylation at residue Ser276 in RAW 264.7 cells was also PKC dependent (Figure 5C). These studies suggested that PKC(s) regulated Ser276 phosphorylation but not Ser536 in both human MDMs and mouse macrophages after M-CSF stimulation.
We next performed cellular fractionation to identify the cellular location of phosphorylated NF-κB p65 in Raw 264.7 cells. Non-phosphorylated NF-κB p65 was located in both cytosolic and nuclear fractions, but phosphorylated Ser276 and Ser536 NF-κB p65 was primarily located in nuclear fraction after M-CSF stimulation (Figure 5D). Notably, constitutive phosphorylation of Ser536 NF-κB p65 was found in these cells. Importantly, Ro-31-8220 reduced M-CSF-induced Ser276 phosphorylation of NF-κB p65 in both the cytosolic and nuclear fractions, while M-CSF-induced NF-κB p65 Ser536 phosphorylation was present in the nucleus regardless of PKC inhibition. These observations indicate that M-CSF-induced Ser276 and Ser536 are regulated differently by conventional PKC activation in mononuclear phagocytes. The purity of the cytosol and nuclear cell fractions was confirmed by immunoblotting with GAPDH and Lamin B, respectively (Figure 5D).