Establishing a new melanoma cell line (PAK1+/ PAK4-) whose melanogenesis depends on PAK1, but not on PAK4.

Such a cell line could be very useful for a highly selective screening for a variety of PAK1-blokcers in cell culture. Since the parental melanoma cell line B16F10 expresses only a minute amount of PAK1, while the expression of PAK4 is very high, PAK1-blockers such as CAPE, FTY720 and pDAP block only upto 50% of melanogenesis even at the highest concentrations. 

However, if we transfect this cell line with  the exogenous Myc-tagged PAK1, its sensitivity to PAK1-blockers would be significantly boosted, because its melanogenesis becomes more dependent on PAK1, and less dependent on PAK4. Furthermore, if we stably transfect this Myc-tagged PAK1 transfectant with PAK4-specific Sh-RNA (ShPAK4), we could basically eliminate the PAK4- dependency of the melanogenesis. 

CAPE (caffeci acid phenethyl ester) down-regulates RAC, but not CDC42. Thus, it selectively blocks the RAC-dependent activation of PAK1, but not the CDC42-dependent activation of PAK4.  Thus, to confirm the exclusive PAK1-dependency of the melanogenesis in this new cell line (PAK+ /PAK4-) , we shall test if CAPE, a typical PAK1-blocker, could inhibit its melanogenesis by almost 100%.  The above project will be one of the first ambitious goals at our newly set-up "PAK Research Center" in Okinawa.