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    • Ivermectin mg br Identification of the kinase that

    2024-03-28


    Identification of the kinase that phosphorylates P450c17 The first Ser/Thr kinase that phosphorylates P450c17 was identified only recently. Of the 518 kinases in the human ‘kinome’ 278 are Ser/Thr kinases (Manning et al., 2002), but microarrays identified only 145 Ser/Thr kinases in human NCI-H295A adrenocortical carcinoma cells (Tee et al., 2008). Several notable kinases were absent, including p70S6K (RPS6KB2), mammalian target of rapamycin (mTOR), phosphatidylinositol 3-kinase (PI3K), MAPK3/ERK1, MAPK1/ERK2, MAP2K1/MEK1, and MAP2K2/MEK2. Thus the pathways involving extracellular signal-regulated kinases 1/2 (ERK 1/2) and mitogen activated protein kinase kinase 1/2 (MEK 1/2), which have been implicated in the insulin resistance of the polycystic ovary syndrome (PCOS) (Corbould et al., 2006) cannot be involved in P450c17 phosphorylation in NCI-H295A cells. Incubations with a panel of kinase inhibitors also appeared to exclude the PKA/PI3K/Akt and calcium/calmodulin/MEK pathways, and suggested a role for rho-associated, coiled-coil containing protein kinase 1 (ROCK1). However, although ROCK1 could phosphorylate P450c17 in vitro, this phosphorylation did not affect 17,20 lyase activity; hence ROCK1 may act upstream of another kinase or act as a scaffolding protein, but its direct phosphorylation of P450c17 is catalytically inconsequential. Of the 145 Ser/Thr kinases in NCI-H295A cells, only six were induced by 8Br-cAMP, and knockdown of each of these by RNA interference had no effect on 17,20 lyase activity (Tee et al., 2008). Studies of P450c17 have been hampered by the difficulty in preparing large amounts of pure, catalytically active protein. Dramatic improvements in bacterial expression of P450c17 were achieved by altering the N-terminal sequence of P450c17 to favor bacterial expression and adding a carboxy-terminal 4x-His tag for affinity purification; however, the purification procedure entails three chromatographic steps on nickel nitrilotriacetic Ivermectin mg (Ni–NTA) agarose, followed by two steps on hydroxylapatite (Imai et al., 1993). We constructed a P450c17 bacterial expression vector with a Gly–Gly-Gly spacer preceding a 6x His tag in the C terminus (G3H6). The glycine residues improve flexibility between the P450c17 moiety and the His tag, which was lengthened from four to six residues to permit more stringent washing conditions. This G3H6 construct permits purification of P450c17 with a single pass on Ni–NTA agarose (Wang et al., 2010); the same C-terminal G3H6 tag strategy also works equally well with POR (Sandee and Miller, 2011) (Fig. 4). Among the cAMP-inducible kinases in NCI-H295A cells was the mitogen-activated protein kinase (MAPK) p38δ (MAPK13) (Tee et al., 2008), which is a major okadaic acid-responsive MAPK and can inactivate ERK1/2 activity directly (Efimova et al., 2003). Therefore we considered the potential roles of the four p38 isoforms: p38α (MAPK14), p38β (MAPK11), p38δ (MAPK13), and p38γ (MAPK12) (Tee and Miller, 2013). Both p38α and p38β have been implicated in regulating steroidogenesis (Inagaki et al, 2009, Nelson-Degrave et al, 2005), and hence seemed to be good candidates. The pyridinyl imidazole drugs SB202190 and SB203580, which inhibit p38α and p38β but not p38δ or p38γ (Cargnello and Roux, 2011) did not affect 17α-hydroxylase activity, but each eliminated detectable 17,20 lyase activity. Co-transfecting non-steroidogenic COS-1 cells with P450c17 and p38α increased 17,20 lyase activity greater than two-fold, but co-transfection of P450c17 with p38β had no effect on 17,20 lyase activity, and neither kinase affected 17α-hydroxylase activity (Fig. 5A). Co-expression of P450c17 and FLAG-tagged p38 isoforms in COS-1 cells showed that p38α associated with P450c17 more avidly than p38β (Tee and Miller, 2013). Bacterially-expressed human P450c17 (Wang et al., 2010) was readily phosphorylated in vitro with p38α (Fig. 5B). This phosphorylation increased 17,20 lyase activity, showing that the effect of p38α resulted from phosphorylation of P450c17 itself, rather than activation of some other cellular kinase: using pure, bacterially-expressed human POR (Sandee and Miller, 2011) as the electron donor, P450c17 that had been phosphorylated by p38α in vitro exhibited 17,20 lyase activity, but non-phosphorylated P450c17 had much less 17,20 lyase activity (Fig. 5C). Knockdown of p38 isoforms with siRNA in human adrenal NCI-H295A cells showed that 17,20 lyase activity was inhibited by knockdown of p38α but not by knockdown of p38β or by a scrambled shRNA control (not shown). Thus p38α augments the 17,20 lyase activity of P450c17 in vivo as well as in vitro. Assessing the enzymology of bacterially-expressed P450c17 with or without phosphorylation by p38α showed that phosphorylation had no effect on the Vmax or Km of the 17α-hydroxylase reaction and that both phospho- and de-phospho P450c17 had similar Km values for the 17,20 lyase reaction, but phosphorylated P450c17 had a significantly greater Vmax (Fig. 5D). This indicates that the phosphorylation did not affect substrate binding, but instead affected the interaction of P450c17 with POR, and hence on substrate turnover.