Biochemical Journal

Research article

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

Gopal P. SAPKOTA, Jérôme BOUDEAU, Maria DEAK, Agnieszka KIELOCH, Nick MORRICE, Dario R. ALESSI

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 Ser431, a previously characterized phosphorylation site, is also phosphorylated at Ser31, Ser325 and Thr366. Incubation of wild-type LKB1, but not a catalytically inactive mutant, with manganese-ATP in vitro resulted in the phosphorylation of LKB1 at Thr336 as well as at Thr366. We were unable to detect autophosphorylation at Thr189, a site previously claimed to be an LKB1 autophosphorylation site. A catalytically inactive mutant of LKB1 was phosphorylated at Ser31 and Ser325 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 Thr336, and perhaps to a lesser extent at Thr366, inhibits LKB1 from suppressing cell growth.

  • cell cycle
  • mass spectrometry
  • phosphopeptide mapping
  • signal transduction

Footnotes

  • Abbreviations used: AMPK, AMP-activated protein kinase; Brg1, brahma-related gene 1 protein; CHK1, checkpoint kinase 1; DMEM, Dulbecco's modified Eagle's medium; ERK, extracellular signal-regulated kinase; GST, glutathione S-transferase; HEK, human embryonic kidney; JNK, c-Jun N-terminal kinase; MALDI—TOF, matrix-assisted laser-desorption ionization—time-of-flight; MAP, mitogen-activated protein; MAPKAP-K2, MAP kinase-activated protein kinase-2; PKA, cAMP-dependent protein kinase; PKB, protein kinase B; S6K, p70 ribosomal S6 kinase; p90RSK, p90 ribosomal S6 kinase; T336-P antibody, phosphospecific antibody recognizing LKB1 phosphorylated at Thr336; T366-P antibody, phosphospecific antibody recognizing LKB1 phosphorylated at Thr366; YFP, yellow fluorescent protein.