Pak3 augments Tax induced activation of HTLV 1 LTR in a kinase independent manner Augmentation of Tax activated LTR activity by group I Paks raised the possibility for suppression of HTLV 1 tran scription with small molecule selleck kinase inhibitor inhibitors of these kinases. As the first step to test this idea, we sought to determine the requirement of the kinase activity of Paks for the stimu latory effect on HTLV 1 LTR. Since Pak1, Pak2 and Pak3 are strikingly homologous, we focused our analysis on Pak3 only. We constructed various mutants of Pak3, some of which have been found in patients with X linked mental re tardation caused by a defect in Pak3. K297L, A365E and R419X mutants are kinase defective, whereas T421E is a dominant active mutant. R67C is a mutant defective of Cdc42 binding.

All mutants were expressed to com parable levels. Several slow migrating bands were noted in the samples of Pak3 wild type as well as R67C and T421E mutants. suggesting that they might be auto phosphorylated. These slow migrating bands disappeared when the samples were treated with calf intestinal phosphatase. For the kinase dead K297L, A365E and R419X mutants, auto phosphorylation Inhibitors,Modulators,Libraries of Pak3 was not observed. To our surprise, all mutants of Pak3 still augmented Tax induced LTR activation equally well and in a dose dependent manner. Hence, kinase or Cdc42 binding activity of Pak3 was not essential for augmentation of Tax Inhibitors,Modulators,Libraries activity on the LTR. We went on to map the Tax augmenting domain of Pak3 using a series of truncated mutants. These mutants were expressed to comparable levels in HeLa cells and interrogated for the ability to potentiate Tax induced activation of the LTR.

All mutants except M4, which contains the CRIB domain alone, were capable of augmenting LTR activation by Tax. These Inhibitors,Modulators,Libraries results indicated that the N terminal regulatory do main was sufficient for augmentation of Tax induced LTR activation. This is generally consistent with our findings obtained from the analysis of Pak3 point mutants. To shed light on the functional Inhibitors,Modulators,Libraries domain of Tax that in teracts with Pak3, we also examined the effect of Pak3 on various point mutants of Tax. Pak3 was found to augment all Tax mutants that are able to activate HTLV 1 LTR. including S258A, S150A, and C23S. Thus, as in the case of many other Tax binding proteins. we were unable to define a discrete domain of Tax that interacts with Pak3.

Plausibly, the interaction is conformation dependent and requires the full protein of Tax. CREB, Inhibitors,Modulators,Libraries CRTCs and p300CBP are required for Tax augmenting effect of Pak3 Tax induced activation of HTLV 1 LTR is mediated through CREB transcription different factor as well as CRTC and p300CBP transcriptional coactivators. Although Pak3 can augment Tax activity on the LTR, it is unclear whether the action of Pak3 would bypass any of these mediators.