Fig. 4. Colchicine stimulates PAI-1 synthesis in quiescent R22 cells. Growth-arrested cells were treated with DMSO (solvent control) or colchicine (10 µM, final concentration) for 4 hours. The cellular microtubule network and PAI-1 protein were visualized in fixed and permeabilized cells by indirect immunocytochemistry with antibodies to tubulin and PAI-1, respectively (A). Cells exposed to DMSO were well spread with a highly branched microtubule network. Colchicine treatment disrupted the microtubule skeleton and significantly reduced the cell-spread area (Fig. 3, legend). PAI-1 protein was virtually undetectable by immunocytochemistry (A) and resolved at only extremely low levels by western blotting of extracts of control cells (B). Abundant PAI-1 protein was easily identified, by contrast, by both techniques in 4 hour colchicine-stimulated cells (A,B). Nuclei were stained with DAPI.

Fig. 5. Metabolic requirements for CD-induced PAI-1 mRNA expression. Serum-deprived cells were pretreated for 30 minutes with actinomycin D (Act D; 5 µg/ml) or puromycin (Puro; 100 µg/ml) prior to a 4 hour exposure to CD (final concentration 10 µM). Optimal concentrations of inhibitors were determined previously (e.g. Ryan et al., 1996{Go}; Hawks and Higgins, 1998{Go}). Northern blots were probed with 32P-labeled cDNAs to PAI-1 and A50 (A). CD-induced PAI-1 transcription appeared to utilize a secondary (i.e. protein-synthesis-dependent) response mechanism. To evaluate the potential role of autocrine TGF-ß as a secondary response intermediate, quiescent R22 cell cultures were pretreated for 30 minutes with TGF-ß neutralizing antibodies prior to addition of either CD or TGF-ß1. RNA was isolated, and blots were probed for PAI-1 and A50 transcripts (B). Representative blots are shown.

Fig. 6. CD-induced PAI-1 expression is sensitive to tyrosine kinase inhibitors. Quiescent R22 cultures were pretreated for 30 minutes with progressively increasing concentrations of genistein (A) or PPI (B) prior to CD treatment. RNA was isolated, and northern blots were probed for PAI-1 and A50 transcripts. Representative blots are shown in A and B. —/— refers to DMSO vehicle only. The corresponding histograms illustrate PAI-1 mRNA abundance normalized to A50 signal for replicate experiments (mean±s.d.). Concentrations of genistine and PP1 (>25 µM and 10 nM, respectively) effectively ablated PAI-1 transcript expression.

Fig. 7. CD activates pp60c-src kinase activity. Cells were incubated in 10 µM CD and extracts prepared at the times indicated for the immune complex assay of pp60c-src activity. Autophosphorylated pp60c-src kinase and phosphorylated IgG heavy chain were evident at 7.5 minutes after CD addition and maximal at 15 minutes post-stimulation (A). Kinase activity rapidly declined thereafter. Addition of PP1 to cultures prior to CD treatment effectively inhibited pp60c-src kinase activity at the optimal 15 minute time point. Equal gel loading was confirmed by visualization of the IgG heavy chain by staining with Ponceau S (B) and by western blotting for pp60c-src and IgG (C). The histogram indicates quantitative data (mean±s.d.) for three independent experiments.

Fig. 8. CD-mediated PAI-1 expression and ERK1/2 activation is MEK dependent. Quiescent R22 cells were pretreated with PD98059 (at the indicated concentrations) prior to addition of CD. Northern blots of total cellular RNA were probed with 32P-labeled cDNA probes to PAI-1 and A50; PAI-1 mRNA abundance was normalized to A50 signal (A). CD-induced ERK1/2 activity was assessed by a coupled immunoprecipitation—in-vitro-kinase assay using myelin basic protein (MBP) as a phosphorylation substrate with or without PD98059 pretreatment (B). Western blotting and Ponceau S staining (B) served to confirm ERK1/2 and MBP levels, respectively, for normalization of MBP phosphorylation activity. The histograms (A,B) illustrate results of three separate experiments (mean±s.d.). Untreated groups in both panels refer to DMSO vehicle only.

Fig. 9. ERK1 and ERK2 are activated by CD treatment. The upper panels in (A,B) illustrate MBP phosphorylation by activated ERK1 and ERK2, respectively, as determined by the coupled immunoprecipitation—in-vitro-kinase assay at various times (mins) following CD addition. PD98059 (20 µM) or serum (final concentration of 20%) was added where indicated. Western blotting and Ponceau S staining served to confirm ERK and MBP levels, respectively (middle and bottom panels in A,B), used for normalization of MBP phosphorylation activity. Data plotted are the means±s.d. for at least three different experiments.

Fig. 10. CD-induced ERK1/2 activity is dependent on a src-like tyrosine kinase. Quiescent R22 cells were preincubated with the indicated inhibitors prior to a 2 hour treatment with CD (10 µM final concentration); control cultures were maintained without additives. Serum-stimulation (FBS to a final concentration of 20%) provided a positive control for ERK activation. The MAPK assay used myelin basic protein (MBP) as the phosphorylation substrate (pMBP; upper panel). Western blot probed with pan-ERK antibodies (middle panel) and Ponceau S staining of MBP (bottom panel) served to confirm equivalent protein loading as well as to confirm normalization of MBP phosphorylation (mean±s.d.) in replicate experiments (histogram).

Fig. 11. Colchicine-induction of PAI-1 expression is sensitive to PP1, puromycin and PD98059 and associated with ERK activation. Pretreatment of quiescent R22 cultures with PP1 significantly attenuated and completely ablated colchicine-mediated PAI-1 expression at 170 and 340 nM, respectively (A, left panel). PAI-1 induction was also effectively inhibited by exposure to puromycin (100 µg/ml) (A, right panel). Similarly, PD98059 at a final concentration of 10 µM reduced cellular PAI-1 levels by 65% whereas use of the inhibitor at 25 µM effectively blocked CD-induced PAI-1 synthesis (B). Exposure of quiescent R22 cells to colchicine-stimulated ERK1/2 phosphorylation by 30 minutes and ERK remained activated for at least 4 hours after addition of the drug (C). ERK phosphorylation, like PAI-1 induction, was PD98059 sensitive. In all cases, colchicine was used at 10 µM.

FIG. 4. Pathway analysis using the Advance Search feature of LiveDIP, applied to the pheromone signaling pathway of yeast. The interactions were generated by the Batch Search feature of LiveDIP, using proteins known to be involved in pheromone signaling transduction (enclosed in the shaded rectangular box) as query molecules (Ste2, Ste18, Ste4, Ste20, Ste5, Ste11, Ste7, Fus3, Dig1, Dig2, and Ste12). The results are represented by the automatically drawn, schematic Interaction Map. The small rectangle boxes depict proteins and complexes, and the lines connecting pairs of boxed proteins represent different kinds of interactions; arrowheads indicate activating interactions, and empty circles indicate inhibiting interactions.