Another notable aspect of this study is that stau rosporine that has been used as an effective inhibitor of various protein kinases, completely inhibited both MAPK like and CDPK activities. It is notewor thy that pretreatments of specific synthetic inhibitors of MAPKs prevented stimulation of the UV B induced MAPK like enzyme activity. however, selleck no effects are observed for the CDPK activity suggest ing that the activation of CDPK was relatively early as compared to the activation of putative MAPK. These data place MAPK downstream intermediaries in the cellular responses mediating catharanthine biosynthesis in response to UV B and position CDPK upstream of MAPK. UV B mediated Tdc Str gene transcription appeared dependent on activation of putative MAPK as well as CDPK pathway.

The activity of a MAPK in cells is control led through phosphorylation activation by its upstream kinases, MAPKK and MAPKKK, and dephosphorylation inactivation by its negative regulator, MAPK phosphatase s. In this study, we showed that the UV B induced MAPK like activity could be inhibited by PD98059, an inhibitor of ERKK, which similar to animal cells has no role to play in UV B signaling. The results obtained using phosphatase inhibitors and NAC should be interpreted with caution because these inhibitors are not specific. NAC, for example is both a free radicle scavenger and phosphatase thiol group protector. Phosphatase inhibitors, on the other hand, can affect the viability of cells at higher concentrations or can mediate an over all up regulation in the kinase activities.

The reason we can attribute to absence of up regulation in any of the UV B induced downstream activities in phosphatase inhibi tors treated cells could probably due to the aberrational or toxic effect of these compounds on the entire cell home ostasis. In fact, treatment of cells with the inhibitors Carfilzomib orthovandate or NAF alone activated many different kinases as assayed by MBP and H IIIS in gel phosphoryla tion assays. The Tdc and Str activity and catharanthine accumulation in orthovanadate or NAF alone treated cells were again comparable to the UV B alone treated cells demonstrating either imbalancing effects on cell homeostasis or that down reg ulation of phosphatases alone are not the only event involved in the up regulation of the TIA pathway and other mechanisms do exist in regulation of TIA biosynthe sis. A tentative model for the UV B signal flows, incorporating these present and previous findings, is illustrated in Figure 9. Upon perception of the UV B light via a putative recep tor, protein phosphorylation is required to induce influx of calcium via plasma membrane channels.