As shown in Figure 7a and 7b, staurosporine, SB 203580, PD 98059 and SB 600125 treatments at the concentrations tested completely abol 17-DMAG structure ished the UV B induced MBPK activity whereas the UV B induced CDPK activity could not be completely inhibited by staurosporine and was not inhibited by SB 203580, PD 98059 and SB 600125 pretreatments of the cells. The inhibitory effect of staurosporine on both MBPK and CDPK activities indicates a common mechanism of action of the inhibitor on these protein kinases, as both of them belong to the family of serine threonine kinases. As expected, inhibitors of the MAPK cascade only inhibited the UV B induced MAPK like MBPK activity, but not CDPK activity. We next examined the accumulation of Tdc and Str mRNAs in protein kinase inhibitor treated cells by reverse transcription polymerase chain reaction.

As shown in Figure 7c staurosporine, SB 203580, PD 98059 and SB 600125 inhibited UV B induced Tdc and Str transcript accumulation. In a similar fashion, UV B induced catharanthine production was significantly decreased by the above mentioned inhibitors indicative of the implication of MBPK and CDPK activities in elicitation of UV B induced catharanthine biosynthesis. The data obtained by immunoprecipitaion experiments and with the use of MAPK cascade specific inhibitors sug gests the involvement of a putative MAPK in response to UV B. As protein phosphatases antagonize the activity of protein kinases, we tested whether pre treatment of cells with pro tein phosphatase inhibitors would show the opposite effect on the UV B induced responses.

Interestingly, the addition of orthovanadate, a known inhibitor of tyrosine phosphatases or sodium fluoride, a compound reported to strongly inhibit serine threonine phos phatases, stimulated only the UV B induced MBPK activity at 1 and 10 mM concentrations substantially above the UV B treated activity while that of CDPK activ ity remained unaffected. The pretreat ment of cells with orthovandate and sodium fluoride did not substantially increase the CDPK activity over and above the UV B treated cells. To further test the role of protein phosphatases in the UV B induced protein phos phorylation activities, we used NAC, which is known to protect the thiol group of phosphatases from inactivation. Pretreatment of cells with NAC inhibited the UV B induced MBPK and CDPK activities at 10 and 100 mM concentrations tested.

As shown in Figure 8c, pretreatment with orthovanadate Cilengitide or NaF did not increase the transcripts of Tdc and Str beyond the levels seen in cells irradiated with UV B alone. however, NAC, on the other hand, decreased the UV B induced accumu lation of Tdc and Str transcripts. At alkaloid level, we found that catharanthine accumulation in the C. roseus cells was greatly increased by UV B irradiation. Pretreatment of orthovanadate or sodium fluoride had no significant effect on the accumulation of catharan thine over and above the cultured C.