Mutations in the LKB1 protein kinase result in the inherited Peutz Jeghers cancer syndrome. LKB1 has been implicated in regulating cell proliferation and polarity although little is known about how this enzyme is regulated. We recently showed that LKB1 is activated through its interaction with STRADa, a catalytically de®cient pseudokinase. Here we show that endogenous LKB1±STRADa complex is associated with a protein of unknown function, termed MO25a, through the interaction of MO25a with the last three residues of STRADa. MO25a and STRADa anchor LKB1 in the cytoplasm, excluding it from the nucleus. Moreover, MO25a enhances the formation of the LKB1±STRADa complex in vivo, stimulating the catalytic activity of LKB1~10-fold. We demonstrate that the related STRADb and MO25b isoforms are also able to stabilize LKB1 in an active complex and that it is possible to isolate complexes of LKB1 bound to STRAD and MO25 isoforms, in which the subunits are present in equimolar amounts. Our results indicate that MO25 may function as a scaffolding component of the LKB1±STRAD complex and plays a crucial role in regulating LKB1 activity and cellular localization.

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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...