Activation of the tumour suppressor kinase LKB1 by the STE20‐like pseudokinase STRAD

The LKB1 gene encodes a serine/threonine kinase mutated in Peutz–Jeghers cancer syndrome. Despite several proposed models for LKB1 function in development and in tumour suppression, the detailed molecular action of LKB1 remains undefined. Here, we report the identification and characterization of an LKB1‐specific adaptor protein and substrate, STRAD (STe20 Related ADaptor). STRAD consists of a STE20‐ like kinase domain, but lacks several residues that are indispensable for intrinsic catalytic activity. Endogenous LKB1 and STRAD form a complex in which STRAD activates LKB1, resulting in phosphorylation of both partners. STRAD determines the subcellular localization of wild‐type, but not mutant LKB1, translocating it from nucleus to cytoplasm. One LKB1 mutation previously identified in a Peutz–Jeghers family that does not compromise its kinase activity is shown here to interfere with LKB1 binding to STRAD, and hence with STRAD‐dependent regulation. Removal of endogenous STRAD by siRNA abrogates the LKB1‐induced G1 arrest. Our results imply that STRAD plays a key role in regulating the tumour suppressor activities of LKB1.

[1]  T. Mäkelä,et al.  Growth suppression by Lkb1 is mediated by a G(1) cell cycle arrest. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[2]  M. Uhler,et al.  LKB1, a novel serine/threonine protein kinase and potential tumour suppressor, is phosphorylated by cAMP-dependent protein kinase (PKA) and prenylated in vivo. , 2000, The Biochemical journal.

[3]  A. M. Johnston,et al.  SPAK, a STE20/SPS1-related kinase that activates the p38 pathway , 2000, Oncogene.

[4]  J. Johnston,et al.  Mutations of Jak-3 gene in patients with autosomal severe combined immune deficiency (SCID) , 1995, Nature.

[5]  Norinobu M. Watanabe,et al.  The Ste20 group kinases as regulators of MAP kinase cascades. , 2001, Trends in cell biology.

[6]  D. Campbell,et al.  Identification of protein phosphorylation sites by a combination of mass spectrometry and solid phase Edman sequencing. , 2002, Journal of biomolecular techniques : JBT.

[7]  A. Ashworth,et al.  LIP1, a cytoplasmic protein functionally linked to the Peutz-Jeghers syndrome kinase LKB1. , 2001, Human molecular genetics.

[8]  Ronald A. DePinho,et al.  Loss of the Lkb1 tumour suppressor provokes intestinal polyposis but resistance to transformation , 2002, Nature.

[9]  J. Nezu,et al.  Peutz-Jeghers syndrome is caused by mutations in a novel serine threoninekinase , 1998, Nature Genetics.

[10]  S. Goodman,et al.  Very high risk of cancer in familial Peutz-Jeghers syndrome. , 2000, Gastroenterology.

[11]  Norinobu M. Watanabe,et al.  Molecular cloning of MINK, a novel member of mammalian GCK family kinases, which is up‐regulated during postnatal mouse cerebral development , 2000, FEBS letters.

[12]  A. Wilks,et al.  Two novel protein-tyrosine kinases, each with a second phosphotransferase-related catalytic domain, define a new class of protein kinase , 1991, Molecular and cellular biology.

[13]  G. Sapkota,et al.  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* , 2001, The Journal of Biological Chemistry.

[14]  F. Kanai,et al.  LKB1 Associates with Brg1 and Is Necessary for Brg1-induced Growth Arrest* , 2001, The Journal of Biological Chemistry.

[15]  Jiahuai Han,et al.  NIK is a new Ste20‐related kinase that binds NCK and MEKK1 and activates the SAPK/JNK cascade via a conserved regulatory domain , 1997, The EMBO journal.

[16]  C. Hanson,et al.  Identification and Characterization of a Novel Ste20/Germinal Center Kinase-related Kinase, Polyploidy-associated Protein Kinase* , 2003, The Journal of Biological Chemistry.

[17]  R. Bernards,et al.  A System for Stable Expression of Short Interfering RNAs in Mammalian Cells , 2002, Science.

[18]  Q. Deveraux,et al.  ILPIP, a Novel Anti-apoptotic Protein That Enhances XIAP-mediated Activation of JNK1 and Protection against Apoptosis* , 2002, The Journal of Biological Chemistry.

[19]  L. Notarangelo,et al.  Complex Effects of Naturally Occurring Mutations in the JAK3 Pseudokinase Domain: Evidence for Interactions between the Kinase and Pseudokinase Domains , 2000, Molecular and Cellular Biology.

[20]  L. Cantley,et al.  The Peutz-Jegher gene product LKB1 is a mediator of p53-dependent cell death. , 2001, Molecular cell.

[21]  Hiroyuki Miyoshi,et al.  Gastrointestinal hamartomatous polyposis in Lkb1 heterozygous knockout mice. , 2002, Cancer research.

[22]  G. Sapkota,et al.  Identification and characterization of four novel phosphorylation sites (Ser 31 , Ser 325 , Thr 336 and Thr 366 ) on LKB1/STK11, the protein kinase mutated in Peutz–Jeghers cancer syndrome , 2022 .

[23]  O. Silvennoinen,et al.  The Pseudokinase Domain Is Required for Suppression of Basal Activity of Jak2 and Jak3 Tyrosine Kinases and for Cytokine-inducible Activation of Signal Transduction* , 2002, The Journal of Biological Chemistry.

[24]  S. Pellegrini,et al.  A dual role for the kinase-like domain of the tyrosine kinase Tyk2 in interferon-alpha signaling. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[25]  T. Mäkelä,et al.  Growth arrest by the LKB1 tumor suppressor: induction of p21(WAF1/CIP1). , 2002, Human molecular genetics.

[26]  M. Stratton,et al.  A serine/threonine kinase gene defective in Peutz–Jeghers syndrome , 1998, Nature.

[27]  J. Nezu,et al.  Loss of cytoplasmic retention ability of mutant LKB1 found in Peutz-Jeghers syndrome patients. , 1999, Biochemical and biophysical research communications.

[28]  Daniel St Johnston,et al.  A role for Drosophila LKB1 in anterior–posterior axis formation and epithelial polarity , 2003, Nature.

[29]  O. Silvennoinen,et al.  Regulation of the Jak2 Tyrosine Kinase by Its Pseudokinase Domain , 2000, Molecular and Cellular Biology.

[30]  D. Morton,et al.  The C. elegans par-4 gene encodes a putative serine-threonine kinase required for establishing embryonic asymmetry. , 2000, Development.

[31]  D. Payan,et al.  TNIK, a Novel Member of the Germinal Center Kinase Family That Activates the c-Jun N-terminal Kinase Pathway and Regulates the Cytoskeleton* , 1999, The Journal of Biological Chemistry.

[32]  J. Nezu,et al.  Peutz-Jeghers syndrome is caused by mutations in a novel serine threonine kinase. , 1998, Nature genetics.

[33]  K. Alitalo,et al.  Vascular Abnormalities and Deregulation of VEGF in Lkb1-Deficient Mice , 2001, Science.

[34]  T. Roberts,et al.  Mutation in the Jak kinase JH2 domain hyperactivates Drosophila and mammalian Jak-Stat pathways , 1997, Molecular and cellular biology.

[35]  A. Villa,et al.  Structural and functional basis for JAK3-deficient severe combined immunodeficiency. , 1997, Blood.

[36]  K. Jishage,et al.  Role of Lkb1, the causative gene of Peutz–Jegher's syndrome, in embryogenesis and polyposis , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[37]  M. Seldin,et al.  Hepatocellular carcinoma caused by loss of heterozygosity in Lkb1 gene knockout mice. , 2002, Cancer research.

[38]  J. Kuriyan,et al.  The Conformational Plasticity of Protein Kinases , 2002, Cell.

[39]  L. Aaltonen,et al.  Induction of cyclooxygenase-2 in a mouse model of Peutz–Jeghers polyposis , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[40]  H Clevers,et al.  The human TCF-1 gene encodes a nuclear DNA-binding protein uniquely expressed in normal and neoplastic T-lineage lymphocytes. , 1995, Blood.