To test whether or not JNK1 directly phosphorylates Negative, purified wt GST JNK1 or kinase deficient GST JNK1 , in which Thr1 and Tyr1 have been replaced by nonphosphorylatable alanines, was modified in an in vitro kinase reaction containing nonradioactive ATP and aliquot of extracts from cells transfected by using a constitutively active MEKK1 , isolated by glutathione Sepharose beads, and extensively washed. Under these disorders, wt GST JNK1 was activated, because it significantly phosphorylated GST c Jun . Meanwhile, the wt GST JNK1 substantially phosphorylated GST Lousy, whilst the kinasedeficient GST JNK1 only includes a minor phosphorylation on GST Lousy . So, these final results strongly indicate that Epoactivated JNK1 is really a Bad kinase Epo activated JNK1 phosphorylation of Bad at Thr21 Our earlier data indicated that phosphorylation of Lousy by JNK1 at threonine 21 contributed to IL mediated cell survival . To test irrespective of whether Epo activated JNK1 could also phosphorylate Terrible at Thr21, basal JNK1 was isolated from Epo deprived HCD cells and lively JNK1 was isolated from Epo stimulated HCD cells. Immunoblotting with anti phospho Thr21 antibody showed that Epo activated JNK1 phosphorylated only wt GST Awful but not the GST Poor mutant . Additionally, in HCD cells, expression with the constitutively lively MKK JNK1 but not the kinase deficient MKK JNK1 resulted in phosphorylation of cotransfected M2 Bad at Thr21 during the absence of Epo .
To more verify Epo activated JNK1 induce Poor phosphorylation at Thr21, HCD cells had been deprived of Epo for one h, and followed by Epo readdition for one min. Immunoblotting with anti phospho Thr21 antibody showed that Terrible was specifically phosphorylated at Thr21 right after Epo readdition . The phosphorylation of Undesirable at Thr21 occurred as early as 1 min right after Epo readdition, corresponding towards the initiation of JNK1 activation JAK3 inhibitor kinase inhibitor by Epo . Phosphorylation of Bad by JNK1 at Thr21 might greatly reduce Bad association with Bcl XL thus inhibiting the pro apoptotic action of Undesirable. To test the Lousy and Bcl XL interaction in response to Epo stimulation, wt GST Bad or mutant GST Awful proteins had been subjected to phosphorylation by Epo activated JNK1 within the presence of nonradioactive ATP. GST pull down assays exposed that phosphorylation by lively JNK1 considerably decreased the binding of wt GST Terrible but not mutant GST Lousy to S labeled Bcl XL .
To additional confirm that Poor phosphorylation at Thr21 regulates the professional apoptotic action SB 271046 kinase inhibitor of Awful, HCD cells stably expressing wt Awful or the Bad mutant had been utilized to determine their susceptibility to Epo withdrawal induced apoptosis. Immunoblotting showed the expression of Poor mutant was equivalent to that of wt Negative . Having said that, cells expressing the Terrible mutant were far more delicate to Epo withdrawal induced apoptosis than cells expressing wt Lousy .