title: Identification and characterization of four novel phosphorylation sites (Ser31, Ser325, Thr336 and Thr366) on LKB1/STK11, the protein kinase mutated in Peutz-Jeghers cancer syndrome.
abstract: Peutz-Jeghers syndrome is an inherited cancer syndrome, which results in a greatly increased risk of developing tumours in those affected. The causative gene encodes a nuclear-localized protein kinase, termed LKB1, which is predicted to function as a tumour suppressor. The mechanism by which LKB1 is regulated in cells is not known, and nor have any of its physiological substrates been identified. Recent studies have demonstrated that LKB1 is phosphorylated in cells. As a first step towards identifying the roles that phosphorylation of LKB1 play, we have mapped the residues that are phosphorylated in human embryonic kidney (HEK)-293 cells, as well as the major in vitro autophosphorylation sites. We demonstrate that LKB1 expressed in HEK-293 cells, in addition to being phosphorylated at Ser(431), a previously characterized phosphorylation site, is also phosphorylated at Ser(31), Ser(325) and Thr(366). Incubation of wild-type LKB1, but not a catalytically inactive mutant, with manganese-ATP in vitro resulted in the phosphorylation of LKB1 at Thr(336) as well as at Thr(366). We were unable to detect autophosphorylation at Thr(189), a site previously claimed to be an LKB1 autophosphorylation site. A catalytically inactive mutant of LKB1 was phosphorylated at Ser(31) and Ser(325) in HEK-293 cells to the same extent as the wild-type enzyme, indicating that LKB1 does not phosphorylate itself at these residues. We show that phosphorylation of LKB1 does not directly affect its nuclear localization or its catalytic activity in vitro, but that its phosphorylation at Thr(336), and perhaps to a lesser extent at Thr(366), inhibits LKB1 from suppressing cell growth.
authors.affiliation: MRC Protein Phosphorylation Unit, MSI/WTB complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, UK. g.p.sapkota@dundee.ac.uk
authors.forename: Gopal P
authors.initials: GP
authors.lastname: Sapkota
authors.forename: Jérôme
authors.initials: J
authors.lastname: Boudeau
authors.forename: Maria
authors.initials: M
authors.lastname: Deak
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Kieloch
authors.forename: Nick
authors.initials: N
authors.lastname: Morrice
authors.forename: Dario R
authors.initials: DR
authors.lastname: Alessi
mesh_terms: D000091162:AMP-Activated Protein Kinase Kinases; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004330:Drosophila; D004622:Embryo, Mammalian; D004625:Embryo, Nonmammalian; D006801:Humans; D007668:Kidney; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010580:Peutz-Jeghers Syndrome / Q000235:genetics*; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism*; D010769:Phosphothreonine / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014162:Transfection
chemical_list: D010769:Phosphothreonine; D010768:Phosphoserine; D017346:Protein Serine-Threonine Kinases; C109790:STK11 protein, human; D000091162:AMP-Activated Protein Kinase Kinases