Phosphorylation of Serine 399 in LKB1 Protein Short Form by Protein Kinase Cξ Is Required for Its Nucleocytoplasmic Transport and Consequent AMP-activated Protein Kinase (AMPK) Activation

Two splice variants of LKB1 exist: LKB1 long form (LKB1L) and LKB1 short form (LKB1S). In a previous study, we demonstrated that phosphorylation of Ser-428/431 (in LKB1L) by protein kinase C xi (PKC xi) was essential for LKB1-mediated activation of AMP-activated protein kinase (AMPK) in response to oxidants or metformin. Paradoxically, LKB1S also activates AMPK although it lacks Ser-428/431. Thus, we hypothesized that LKB1S contained additional phosphorylation sites important in AMPK activation. Truncation analysis and site-directed mutagenesis were used to identify putative PKC xi phosphorylation sites in LKB1S. Substitution of Ser-399 to alanine did not alter the activity of LKB1S, but abolished peroxynitrite-and metformin-induced activation of AMPK. Furthermore, the phosphomimetic mutation (S399D) increased the phosphorylation of AMPK and its downstream target phospho-acetyl-coenzyme A carboxylase (ACC). PKC xi-dependent phosphorylation of Ser-399 triggered nucleocytoplasmic translocation of LKB1S in response to metformin or peroxynitrite treatment. This effect was ablated by pharmacological and genetic inhibition of PKC xi, by inhibition of CRM1 activity and by substituting Ser-399 with alanine (S399A). Overexpression of PKC xi up-regulated metformin-mediated phosphorylation of both AMPK (Thr-172) and ACC (Ser-79), but the effect was ablated in the S399A mutant. We conclude that, similar to Ser-428/431 (in LKB1L), Ser-399 (in LKB1S) is a PKC xi-dependent phosphorylation site essential for nucleocytoplasmic export of LKB1S and consequent AMPK activation.