Inhibition of p38 MAPK inhibits translocation of FGF1 to the cytosol and nucleus. (A) In vivo phosphorylation of exogenously added FGF1 in NIH 3T3 cells was analyzed in the absence or presence of micromolar concentrations of SB203580 or 10 nM bafilomycin A1 (Baf.A1), as indicated. In vivo-phosphorylated FGF1 (³³P-FGF1) was detected by fluorography, and the total cellular uptake of FGF1 (total FGF1) was detected by anti-FGF1 immunodetection. (B) NIH 3T3 cells were incubated with or without 5 μM anisomycin in the absence or presence of micromolar concentrations of SB203580 as indicated. Total cell lysates were analyzed by Western blotting using an anti-phospho-MK2-specific antibody (p-MK2) and an anti-total MK2 antibody. (C) The in vivo phosphorylation of FGF1 was analyzed in the presence of micromolar concentrations of PD169316 or SB202474 or 10 nM bafilomycin A1 as indicated. (D) In vivo phosphorylation of FGF1 was analyzed in the absence or presence of 5 μM SB203580, 20 nM TPA, or 10 nM bafilomycin A1 as indicated. (E) NIH 3T3 cells were incubated with in vitro-labeled ³⁵S-FGF1 and heparin for 6 h in the absence or presence of micromolar concentrations of SB203580 or 10 nM bafilomycin A1 as indicated. The cells were fractionated into membrane (M), cytosolic (C), and nuclear (N) fractions. FGF1 was extracted from each fraction by binding to heparin-Sepharose and analyzed by SDS-PAGE and fluorography. (C and N fractions were exposed to film four times longer than the M fractions.) (F) The subcellular fractions obtained by fractionation as for panel E were tested for the presence of the membrane-associated proteins Rab5a and calreticulin, the cytosolic protein ERK1/2, and the nuclear protein lamin A by specific antibodies.

Activation of p38 MAPK by anisomycin enhances translocation of FGF1. (A) In vivo phosphorylation of exogenously added FGF1 in NIH 3T3 cells was analyzed in the presence of increasing micromolar concentrations of anisomycin or 10 nM bafilomycin A1 (Baf.A1) as indicated. In vivo phosphorylated FGF1 (³³P-FGF1) was detected by fluorography, and the total cellular uptake of FGF1 (total FGF1) was detected by anti-FGF1 immunodetection. (B) NIH 3T3 cells were incubated with in vitro-labeled ³⁵S-FGF1 and heparin for 6 h in the presence of increasing concentrations of anisomycin as indicated. The cells were fractionated into membrane (M), cytosolic (C), and nuclear (N) fractions. FGF1 was extracted from each fraction by binding to heparin-Sepharose and analyzed by SDS-PAGE and fluorography. (C and N fractions were exposed to film four times longer than the M fractions.) (C) In vivo phosphorylation of FGF1 was analyzed in the absence or presence of 10 μM anisomycin, 1 μM Gö6976, 5 μM rottlerin, or 10 nM bafilomycin A1 as indicated. (D) NIH 3T3 cells were treated as explained for panel B but in the absence or presence of 10 μM anisomycin, 1 μM Gö6976, or 5 μM rottlerin as indicated. (E) The activity of PKCδ in the presence of various inhibitors was tested in vitro. Lysates of NIH 3T3 were subjected to immunoprecipitation (IP) using anti-Sumo-1 antibody (ctrl; lane 1) or anti-PKCδ antibody (lanes 2 to 7) in the absence (lane1 to 6) or presence (lane 7) of anti-PKCδ antibody-blocking peptide. The immunoprecipitated material was incubated in kinase buffer with MBP and [γ-³³P]ATP, in the presence or absence of 10 μM SB203580, 10 μM anisomycin, 10 μM PD169316, 1 μM Gö6976, or 5 μM rottlerin, and thereafter the samples were analyzed by SDS-PAGE, fluorography (³³P-MBP), and Western blotting using anti-MBP antibody (total MBP).

Effect of activation of p38 MAPK by anisomycin, thapsigargin, mannitol, and H₂O₂ on translocation of FGF1. (A) In vivo phosphorylation of exogenously added FGF1 in NIH 3T3 cells was analyzed in the absence or presence of 10 μM anisomycin, 1 μg/ml thapsigargin, 0.5 M mannitol, 250 μM H₂O₂, or 10 nM bafilomycin A1 (Baf.A1) as indicated. In vivo-phosphorylated FGF1 (³³P-FGF1) was detected by fluorography, and the total cellular uptake of FGF1 (total FGF1) was detected by anti-FGF1 immunodetection. (B) NIH 3T3 cells were treated with 10 μM anisomycin, 1 μg/ml thapsigargin, 0.5 M mannitol, or 250 μM H₂O₂ for 0, 15, or 30 min as indicated. Cell lysates were analyzed by SDS-PAGE and Western blotting using antibodies specific for phosphorylated p38 MAPK (p-p38), total p38 MAPK (p38), phosphorylated MK2 (p-MK2), and total MK2 as indicated.

siRNA knockdown of p38α inhibits translocation of FGF1. (A to C) NIH 3T3 cells were transfected with control siRNA (ctrl) or three different siRNAs specific for mouse p38α (α₁, α₂, and α₃). (A) Total cell lysates were analyzed for p38α (i) and total p38 MAPK (ii) by Western blotting. In vivo phosphorylation of FGF1 was analyzed in the siRNA-transfected cells. Phosphorylated FGF1 (³³P-FGF1) was detected by fluorography (iii), and the total cellular uptake was detected by anti-FGF1 immunodetection (total FGF1 [iv]). (B) In vivo phosphorylation of FGF1 was analyzed in the siRNA-transfected cells in the presence of 10 μM anisomycin. (C) In vivo phosphorylation of FGF1 was analyzed in the siRNA-transfected cells in the absence (i) or presence (ii to v) of LMB, and the cells were fractionated into nuclear (N) and cytoplasmic (C) fractions before extraction of FGF1. (D) Nuclear and cytoplasmic fractions of NIH 3T3 cells obtained by the cellular fractionation method described for panel C were tested for the presence of the cytoplasmic proteins rab5a, calreticulin, and ERK1/2 and the nuclear protein lamin A by specific antibodies. (E and F) BJ cells were mock transfected or transfected with control siRNA (ctrl), two different siRNAs specific for human p38α (α1 and α2), or two different siRNAs specific for human p38β (β1 and β2). (E) The transfected cells were lysed and tested for the amount of p38α (i), p38β (ii), and ERK1/2 (loading control, [iii]) by immunoblotting. Translocation of FGF1 was tested in the transfected cells in an in vivo FGF1 phosphorylation assay. Phosphorylated FGF1 (³³P-FGF1) was detected by fluorography (iv), and the total cellular uptake was detected by anti-FGF1 immunodetection (total FGF1 [v]). No FGF1 was added in the first lane, panels iv and v. (F) siRNA-transfected BJ cells were incubated with in vitro-labeled ³⁵S-FGF1 and heparin for 6 h, and then the cells were fractionated into membrane (M), cytosolic (C), and nuclear (N) fractions. FGF1 was extracted from each fraction by binding to heparin-Sepharose and analyzed by SDS-PAGE and fluorography. (C and N fractions were exposed to film four times longer than the M fractions.) (G) The subcellular fractions obtained by fractionation as described for panel F were tested for the presence of the membrane-associated proteins Rab5a and calreticulin, the cytosolic protein ERK1/2, and the nuclear protein lamin A by specific antibodies.

Effect of serum starvation and FGF1 stimulation on the activity of p38 MAPK. (A) NIH 3T3 cells were incubated for the indicated length of time in serum-free medium. In one case the cells were starved for 24 h and thereafter stimulated with FGF1 and heparin for 15 min. Total cell lysates were analyzed for phosphorylated p38 MAPK (p-p38) and total p38 MAPK by Western blotting. (B) NIH 3T3 cells were serum starved for 24 h and then stimulated with FGF1 and heparin for the indicated periods of time. Total cell lysates were analyzed for phosphorylated p38 MAPK (p-p38), total p38 MAPK (p38), phosphorylated MK2 (p-MK2), and total MK2 by Western blotting, as indicated. (C) In vivo phosphorylation of exogenously added FGF1 in NIH 3T3 cells was analyzed after incubation of the cells with FGF1 for the indicated length of time. In vivo-phosphorylated FGF1 (³³P-FGF1) was detected by fluorography, and the total cellular uptake of FGF1 (total FGF1) was detected by anti-FGF1 immunodetection.

Effect of activation and inhibition of p38 MAPK on endocytosis and intracellular sorting of FGF1. (A) NIH 3T3 cells were incubated with SB203580, SB202474, anisomycin, or PD169316 and increasing concentrations of ¹²⁵I-labeled FGF1 for 3 h at 4°C. The cells were washed with 1 M NaCl and lysed, and the solubilized radioactivity, representing the cell surface FGFR-bound ¹²⁵I-FGF1, was measured (reported as counts per minute [CPM]). (B) HeLa cells transfected with FGFR1 or FGFR4 were incubated with Cy3-FGF1 and heparin in the presence or absence of SB203580 (10 μM) or anisomycin (10 μM) for 2 h at 37°C in DMEM with 0.3 mM leupeptin. The cells were fixed, stained for LAMP-1 (Cy2), and examined by confocal microscopy. Bar, 5 μm. Images shown are merged images (red plus green) where a section of each image has been magnified and shown to the left as red, green, and merged images separately. The proportion of Cy3-positive structures that colocalized with Cy2-positive structures (indicated by yellow in the merged images) was calculated and presented as the mean from 15 cells in each case. Error bars indicate the standard deviations. (C) NIH 3T3 cells were preincubated with or without 20 μM anisomycin for 15 min at 37°C and then incubated with ³⁵S-FGF1 and heparin at 4°C for 2 h. The cells were washed with 1 M NaCl and lysed to measure surface-bound FGF1 or, to measure endocytosis, incubated further for 0, 15, 30, or 60 min at 37°C in the presence and absence of 20 μM anisomycin. ³⁵S-FGF1 was extracted from cell lysates and subjected to SDS-PAGE and fluorography. *, partially degraded FGF1.

Effects of inhibition of p38 MAPK and mutations in FGFR1 on translocation of FGF1 in COS-1 cells. (A) Amino acid sequence of the Ct of FGFR1. The start of exon 17 is marked by an arrow, and Ser777 is marked by an asterisk. (B) In vivo phosphorylation of exogenously added FGF1 in COS-1 cells transfected to express FGFR1 was analyzed in the absence or presence of TPA (20 nM) or micromolar concentrations of SB203580. In vivo-phosphorylated FGF1 (³³P-FGF1) was detected by fluorography, and the total cellular uptake of FGF1 (total FGF1) was detected by anti-FGF1 immunodetection. (C) COS-1 cells were transfected with wild-type FGFR1 or with FGFR1 with point mutations, as indicated, and then the in vivo phosphorylation of FGF1 (³³P-FGF1) and the total cellular uptake (total FGF1) were analyzed. (D and E) COS-1 cells were transfected with wild-type FGFR1 or FGFR1 S777D, and then the in vivo phosphorylation of FGF1 (³³P-FGF1) and the total cellular uptake (total FGF1) was analyzed in the absence or presence of 5 μM SB203580 (D) and in the absence or presence of 5 μM anisomycin and 5 μM SB203580 as indicated (E).

Inhibitory effect of increasing concentrations of SB203580 and wortmannin on translocation of FGF1 in COS-1 cells expressing wild-type FGFR1 or FGFR1 S777D. COS-1 cells were transfected with wild-type FGFR1 or FGFR1 S777D, and then the in vivo phosphorylation of FGF1 was analyzed in the presence of increasing concentrations of SB203580 (A) or wortmannin (B) as indicated. In vivo-phosphorylated FGF1 (³³P-FGF1) was detected by fluorography, and the total cellular uptake of FGF1 (total FGF1) was detected by anti-FGF1 immunodetection.

In vitro phosphorylation of the FGFR1 Ct by recombinant p38α. (A) Recombinant, active (+), or inactive (x) p38α kinase and recombinant fusion proteins of GST and the Ct of wild-type FGFR1 (GST-R1-Ct), FGFR1 S777A (GST-R1-S777A-Ct), or FGFR1 S777D (GST-R1-S777D-Ct) were incubated with [γ-³³P]ATP in reaction buffer at 30°C for 30 min in the presence or absence of 5 μM SB203580. The proteins were analyzed by SDS-PAGE, electroblotting, and autoradiography (upper panel), and then the membrane was stained with Coomassie (lower panel). The 31-kDa band indicates the fusion proteins. (B) In vitro phosphorylation of GST-R1-Ct was performed as for panel A, but in the presence of increasing concentrations of SB203580, as indicated.