Figure 1.
The effect of PKC inhibitors on CSF-1 dependent mitogenesis.
(A) PKC immunoblots. “+” refers to the presence of a competing peptide. Arrow points to the PKC band that is competed off by the corresponding peptide. The lower band in the PKCζ blot is the authentic PKCζ protein (see text). (B) MTS assay. 32D.R cells were plated under the conditions shown and in the presence of 5 nM CSF-1. MTS activity was assayed after 48 h. (C) MTS assay was performed as described in (A) in the presence of the indicated amounts of PKCζ pseudosubstrate peptide. (D) MTS assay in the presence of CSF-1 and the indicated inhibitors, 10 µM U0126 and 5 µM LY294002.
Figure 2.
The effect of PKC inhibitors on CSF-1 mediated Erk, MEK, Raf and Ras activation.
(A, B) 32D.R cells were pretreated with the indicated amounts of inhibitors (Inh) followed by CSF-1 stimulation for 4 min. Lysates were immunoblotted with phospho-Erk (top) or total Erk1/2 (bottom) antibody (A) or with phospho-MEK (top) and total MEK1 (bottom) antibody (B). (C) Cells were processed as described for (A, B). Inhibitors were added at 10 µM. Lysates were immunoprecipitated with either A-Raf or Raf-1 antibody and immune complexes used in an in vitro kinase (IVK) assay with KD-MEK as substrate. Phospho-MEK was then detected by immunoblotting. In (A–C), changes in phosphorylation are expressed as percentages of CSF-1 mediated increase in phosphorylation over unstimulated cells, where 0% and 100% denote phosphorylation in the absence or presence of CSF-1 without inhibitors. (D) Ras activation. Cells were starved and pretreated as indicated with Ro-31-8220 (10 µM or 30 µM) before stimulation with 5 nM CSF-1 for 4 min. Ras-GTP was extracted from 500 µg of lysates using the GST-RBD pull-down assay. 50 µg of total cell lysates were also loaded for comparison.
Figure 3.
CSF-1-stimulated ERK activity is independent of phorbol ester-sensitive PKCs.
(A) 32D.R cells were pretreated with 10 µM GF109203X followed by 5 nM CSF-1 or 0.5 µM PMA stimulation for 4 min. Erk and MEK activity were determined in an in vitro kinase assay with myelin basic protein (MBP) and KD-Erk as substrate respectively. (B) Exponentially growing 32D.R cells were treated with 100 nM PMA for 24 hr to downregulate phorbol ester sensitive-PKCs and continued during the starvation period (designated as *PMA). Cells were either left untreated or treated with 5 nM CSF-1 or 0.5 µM PMA for 4 min. An MBP kinase assay on Erk immunoprecipitates was performed. In (A) and (B), change in phosphorylation is calculated as described in Figure 2. (C) Phorbol ester-sensitive PKCs in 32D.R. Total cell lysates were Western blotted with the indicated anti-PKC antibodies in the absence (−) or presence (+) of the immunizing peptide.
Figure 4.
CSF-1 increases PKCζ activity but does not induce PKCε membrane translocation.
(A) Subcellular fractionation. 32D.R cells were starved, pretreated or not with wortmannin (200 nM) or Ro-31-8220 (30 µM) before stimulation with 5 nM CSF-1 or 0.5 µM PMA for 4 min. Homogenates were separated into cytosolic (S100) and particulate (P100) fractions as described in Methods. 75 µg of each fraction was analyzed by Western blotting with anti-PKCε or PKCζ antibodies. Asterisk indicates the PKCζ protein. (B) CSF-1 stimulated PKCζ kinase activity in an in vitro kinase assay. Ro-31-8220 was included in parallel experiments. (C) CSF-1 stimulated PKCζ phosphorylation. Lysates from 32D.R cells treated as indicated were immunoprecipitated with an anti- PKCζ antibody and blotted with a phospho- PKCζ-T410 antibody. Phosphorylation changes are calculated as described in Figure 2. For reference, at 4 min, CSF-1 stimulated a 7-fold increase in T410 phosphorylation.
Figure 5.
The effect of dominant-negative and constitutively active PKCζ on ERK activation.
(A) Dominant-negative PKCζ. 32D-CSF-1R cells were transfected with 4 µg of HA-ERK and 10 µg of either pcDNA or PKCζ (T/A)4. 24 hr later, cells were starved and stimulated with 10 nM CSF-1 or 0.5 µM PMA for 4 min. Expression of transfected proteins was confirmed by blotting with anti- PKCζ (top) or anti-HA (middle) antibodies. HA immunoprecipitates were analyzed for MBP kinase activity (bottom). Phosphorylation changes are calculated as described in Figure 2. In the experiment shown, CSF-1 induced a 3.9-fold increase in MBP phosphorylation over unstimulated cells. Panel on the right shows the averaged results from 3 independent transfections. Data are expressed as the mean ± SD. (B) Constitutively active PKCζ. Cells were transfected with 10 µg of Myc-ERK and 40 µg of either pcDNA, HA-WT-PKCζ or HA-PKCζcat. They were treated as described in (A). (left) PKCζ in vitro kinase activity. PKCζ was immunoprecipitated using an HA antibody and subjected to an in vitro kinase activity with PKCε pseudosubstrate peptide as a substrate. Shown are the means ± SD (n = 3). (right) Lysates containing approximately equivalent amounts of Myc-ERK were analyzed for MBP kinase activity in anti-Myc immunoprecipitates. Basal MBP phosphorylation in cells transfected with PKCζcat was 1.8 fold over that in cells transfected with an empty vector.
Figure 6.
CSF-1 does not activate an NF-κB luciferase reporter in 32D.R cells.
A 32D.R line stably expressing an NF-κB luciferase reporter was treated as indicated and lysates subjected to a luciferase assay at 6 h or 20 h after stimulation. CSF-1 was used at 5 nM and TNFα at 20 ng/mL. Inhibitors were used at GF109203X (10 µM), Ro-3108220 (1 µM) and Gö 6893 (5 µM).
Figure 7.
WT-PKCζ overexpression enhances CSF-1 dependent proliferation and Erk phosphorylation.
(A) (left) PKCζ protein expression in 32D.R cells transfected with empty vector (pEF) or WT- PKCζ. (right) CSF-1 dose responsive MTS assay. The two curves were statistically different. CSF-1 concentrations were in nM. (B) Erk phosphorylation in control 32D.R cells (C) or 32D.R cells overexpressing WT- PKCζ (WT) in response to CSF-1 stimulation for the indicated times. Lysates were immunoblotted with anti-phospho-Erk (top) or total Erk (bottom) antibodies. Phosphorylation changes are calculated as described in Figure 2 relative to that determined for control cells stimulated by CSF-1 for 1 min (fold increase is 4.6).
Figure 8.
The effect of PKC inhibitors on MEK-Erk activation in bone marrow derived macrophages.
(A) BMMs were starved in the absence of CSF-1 for 12 h, inhibitors were added or not for 1 h followed by stimulation with 5 nM CSF-1. Inhibitor concentrations used were: GF109203X (GF) at 0.1, 1 and 10 µM; Go 6983 (Go) at 0.1, 1 µM and PKCζ-PS peptide at 10 µM. Lysates were immunoblotted with phospho-Erk and total Erk antibodies (top 2 panels) or with phospho-MEK and total MEK antibodies (bottom 2 panels). Phosphorylation changes are shown as a percent of that determined for CSF-1 stimulation at 5 min in the absence of inhibitors. For the data shown, CSF-1 stimulated MEK and Erk by 46- and 62-fold over untreated cells. (B) BMMs were starved in serum free media for 6 h, 1 or 10 µM GF109203X was added as shown followed by CSF-1 stimulation for 5 min. (C) PKCζ phosphorylation. (Top) PKCζ was immunoprecipitated with a rabbit polyclonal antibody and blotted with an antibody that recognizes PKCζ phospho-Thr 410. (Bottom) The blot was stripped and reprobed with a monoclonal antibody that recognizes PKCζ. Shown are representative results from one of two experiments performed. Immunoblot images in Figure 8 were captured digitally using the AlphaImager Gel Imaging System (FluorChem Q, Cell Biosciences, Santa Clara, CA).
Figure 9.
The effect of PKC inhibitors on bone marrow derived macrophage proliferation.
(A) BMMs were treated with the inhibitors indicated in the presence of 5 nM CSF-1 and MTS activity assayed at 48 h, 72 h and 96 h. Similar results were obtained with 0.25 nM and 1 nM CSF-1 (not shown). (B) BMMs were treated with PKCζ PS peptide inhibitor in the presence of CSF-1 and cell counts determined at 96 h. (C) The experiment was performed as described in (A). The concentrations shown are in µM. U0126, 10 µM; LY, 5 µM LY294002.