Phosphorylation of the Protein Kinase Mutated in Peutz-Jeghers Cancer Syndrome, LKB1/STK11, at Ser431 by p90RSK and cAMP-dependent Protein Kinase, but Not Its Farnesylation at Cys433, Is Essential for LKB1 to Suppress Cell Growth

Abstract

did not alter the activity of LKB1 to phosphorylate itself or the tumor suppressor protein p53 or alter the amount of LKB1 associated with cell membranes. The reintroduction of wild-type LKB1 into a cancer cell line that lacks LKB1 suppressed growth, but mutants of LKB1 in which Ser 431 was mutated to Ala to prevent phosphorylation of LKB1 were ineffective in inhibiting growth. In contrast, a mutant of LKB1 that cannot be prenylated was still able to suppress the growth of cells.Peutz-Jeghers syndrome is an autosomal dominantly inherited disorder that predisposes to a wide spectrum of benign and malignant tumors (1, 2). It is caused by mutation of a widely expressed protein kinase of unknown function termed LKB1 (also known as STK11) (3, 4). To date, over 60 different mutations have been mapped to LKB1, many of which would be expected to impair LKB1 activity. These discoveries have aroused great interest because they indicate that LKB1 is likely to function in cells as a tumor suppressor, and consistent with this, overexpression of LKB1 in a number of tumor cell lines has been shown to suppress cell growth by inducing a G 1 cell cycle block (5). However, little is known regarding the mechanism by which LKB1 activity is regulated in cells, and no substrates for LKB1 have thus far been identified.LKB1 is a 436-amino acid protein possessing a kinase domain (residues 50 -337) that is only distantly related to other mammalian kinases. The N-terminal non-catalytic domain comprises both a nuclear localization signal (6) and a putative cytoplasmic retention signal (7). There are no yeast homologs of LKB1, but there are putative homologs in Xenopus (termed XEEK1, with 84% overall identity to LKB1) (8) and Caenorhabditis elegans (termed PAR-4, with 26% overall identity to LKB1 and 41% identity in the kinase domain) (9). In Drosophila, an uncharacterized protein kinase listed in the NCBI Protein Database (NCBI accession number AAF54972) possesses 44% overall identity to LKB1.Recently, a C-terminal fragment of LKB1 was shown to be phosphorylated at Ser 431 by the cAMP-dependent protein kinase (10). Ser 431 of LKB1 lies in the sequence Lys-Xaa-ArgArg-Xaa-Ser (where Xaa is any amino acid), which is conserved in all known mammalian LKB1 sequences and in Xenopus XEEK1. This study did not establish whether full-length or endogenously expressed LKB1 was phosphorylated at Ser 431 in response to stimuli that activated cAMP-dependent protein kinase (PKA) 1 or the role that this phosphorylation played in enabling LKB1 to suppress cell growth. Ser 431 lies in the consensus sequence for phosphorylation by a group of kinases related to PKA, viz. p90 ribosomal S6 kinase (p90 RSK ), mitogen-and stress-stimulated protein kinase (MSK1), and p70 ribosomal S6 kinase (S6K1) (11)(12)(13), that collectively belong to the AGC kinase subfamily. p90 RSK is activated in cells by growth factors and phorbol esters and by ERK1/2 MAPK family members (14), whereas MSK1 is activated in vivo by two dif-* The costs of publication of this article were defr...

Clinicopathologic and Molecular Features of Pancreatic Adenocarcinoma Associated with Peutz-Jeghers Syndrome

“…Consistent with this hypothesis, overexpression of STK11/LKB1 in cultured cells suppresses cell growth, but only in those cell lines with reduced levels of STK11/LKB1 mRNA or those in which STK11/LKB1 kinase activity is impaired [120][121][122] These data imply that STK11/LKB1 is necessary but not sufficient to inhibit cell proliferation, a finding that is consistent with the fact that STK11/LKB1 is expressed in normal proliferating cells such as small intestinal epithelia. 97 Recent data suggests that STK11/LKB1 suppresses cell proliferation by mediating G 1 cell cycle arrest through induction of the cyclindependent kinase inhibitor p21 WAF1/CIP1 in a p53-dependent manner.…”

“…Such modification can be accomplished by epidermal growth factor-activated p90 ribosomal S6 kinase (p90RSK) or forskolin-activated cAMP-dependent protein kinase (PKA). 121 On the other hand, STK11/LKB1 autophosphorylates at threonine-336, and this event in turn prevents itself from exerting growth-suppressive activity. 122 Activation of ataxia telangiectasia mutated kinase (ATM) mediated phosphorylation of STK11/LKB1 at threonine-366 following exposure of cells to ionizing radiation has also been reported.…”

“…Recent studies have indicated that STK11/LKB1 functions as a tumor suppressor by inducing apoptosis and suppressing cell proliferation. 97,120,121 These functions require a kinase activity that is regulated by phosphorylation at threonine and serine sites within the STK11/LKB1 protein. [121][122][123] Current models suggests that the tumor suppressive activities of STK11/LKB1 are mediated through interactions with other tumor suppressor proteins [124][125] or chromatin remodeling molecules 126 (Fig.…”

JNK pathway mediates apoptotic cell death induced by tumor suppressor LKB1 in Drosophila

“…19 Another tumor suppressor p53, the first identified in vitro substrate of LKB1, was proposed as a mediator of this apoptosis. 19,20 Finally, LKB1 was shown to be necessary for the polarization of intestinal epithelial cells and Drosophila ovary cells, demonstrating another important function of LKB1 in regulating cell structure. 21,22 Although considerable progress has been made to characterize the in vivo function of LKB1, only a limited amount of information concerning its molecular mechanisms has been found in detail; the major biological pathway responsible for the tumor suppressive function of LKB1 remains to be clarified.…”

Protein phosphatase 5 modulates SMAD3 function in the transforming growth factor‐β pathway

“…Sodium pyruvate (1X) and non-essential amino acids (0.1mM) were supplemented for Neuro2A, G361 and U87 cells. Mouse ES cells (29) and RAW macrophage cells (27) were maintained as described. The transfection of cDNA vectors was performed as described previously (3).…”