Disruption of 3-phosphoinositide-dependent kinase 1 (PDK1) signaling by the anti-tumorigenic and anti-proliferative agent N-alpha-tosyl-1-phenylalanyl chloromethyl ketone

The anti-tumorigenic and anti-proliferative effects of N-alpha -tosyl-L-phe nylalanyl chloromethyl ketone (TPCK) have been known for more than three de cades. Yet little is known about the discrete cellular targets of TPCK cont rolling these effects. Previous work from our laboratory showed TPCK, like the immunosuppressant rapamycin, to be a potent inhibitor of the 70-kilodal ton ribosomal S6 kinase 1 (S6K1), which mediates events involved in cell gr owth and proliferation. We show here that rapamycin and TPCK display distin ct inhibitory mechanisms on S6K1 as a rapamycin-resistant form of S6K1 was TPCK-sensitive, Additionally, we show that TPCK inhibited the activation of the related kinase and proto-oncogene Akt. Upstream regulators of S6K1 and Akt include phosphoinositide S-kinase (PI 3-K) and 3-phosphoinositide-depe ndent kinase 1 (PDK1). Whereas TPCK had no effect on either mitogen-regulat ed PI 3-K; activity or total cellular PDK1 activity, TPCK prevented phospho rylation of the PDK1 regulatory sites in S6K1 and Akt. Furthermore, whereas both PDK1 and the mitogen-activated protein kinase (MAPK) are required for full activation of the 90-kilodalton ribosomal S6 kinase (RSK), TPCK inhib ited RSK activation without inhibiting MAPK activation. Consistent with the capacity of RSK and Abt to mediate a cell survival signal, in part through phosphorylation of the pro-apoptotic protein BAD, TPCK reduced BAD phospho rylation and led to cell death in interleukin-3-dependent 32D cells. Finall y, in agreement with results seen in embryonic stem cells lacking PDK1, pro tein kinase A activation was not inhibited by TPCK showing TPCK specificity for mitogen-regulated PDK1 signaling. TPCK inhibition of PDK1 signaling th us disables central kinase cascades governing diverse cellular processes in cluding proliferation and survival and provides an explanation for its stri king biological effects.