ABSTRACT
BACKGROUND: Ksr (kinase supressor of Ras) was identified as a regulator of the Ras-MAP kinase (mitogen-activated protein kinase) pathway by genetic screens in Drosophila and Caenorhabditis elegans. Ksr is a kinase with similarities to the three conserved regions of Raf kinases, especially within the kinase domain. To investigate whether these structural similarities correlated with common functional properties, we examined the ability of mKsr-1, the murine homolog of Ksr, to interact with components of the vertebrate MAP kinase pathway. RESULTS: In the yeast two-hybrid interaction assay, mKsr-1 did not bind to either Ras, B-Raf or Raf-1, but interacted strongly with both MEK-1 and MEK-2, activators of MAP kinase. The Ksr-MEK interaction was confirmed by co-immunoprecipitation experiments. Ectopically expressed mKsr-1 co-precipitated with endogenous MEK-1 in COS-1 cells, and endogenous Ksr and MEK co-precipitated from PC12 cells. Phosphorylation of MEK by mKsr-1 was not detected, however. In contrast, the MEK subpopulation complexed with mKsr-1 in COS-1 cells or PC12 cells did not display kinase activity. This ability of Ksr to block MEK in an inactive form correlated with a biological response: mKsr-1 did not transform NIH3T3 cells, and, furthermore, mKsr-1 reduced Ras-induced transformation. Similarly, mKsr-1 inhibited the proliferation of embryonic neuroretina cells induced by Ras and B-Raf but not that induced by MEK. CONCLUSIONS: Our results suggest a novel mechanism for Ksr in regulating the MAP kinase pathway, at least in part through an ability to interact with MEK.
Subject(s)
Cell Transformation, Neoplastic/drug effects , Mitogen-Activated Protein Kinase Kinases , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Signal Transduction , ras Proteins/antagonists & inhibitors , 3T3 Cells , Animals , COS Cells , Cell Division/drug effects , Chick Embryo , Epidermal Growth Factor/pharmacology , MAP Kinase Kinase 1 , MAP Kinase Kinase 2 , Mice , Nerve Growth Factors/pharmacology , PC12 Cells , Proto-Oncogene Proteins c-raf/metabolism , Rats , Retina/drug effectsABSTRACT
The LAMMER subfamily of kinases has been conserved throughout evolution, and its members are thought to play important roles in the regulation of cellular growth and differentiation programs. STY is a murine LAMMER kinase which has been implicated in the control of PC12 cell differentiation. Multiple transcripts are derived from the Sty gene, and their relative abundance is developmentally regulated. Alternative splicing of the primary Sty transcript generates mRNAs encoding full-length catalytically active (STY) and truncated, kinase-deficient polypeptides. Both STY and its truncated isoform, STYT, are localized in the nucleus and are capable of heterodimerizing. We also demonstrate that STY functions as a dual specificity kinase in mammalian cells.
Subject(s)
Alternative Splicing , Cell Nucleus/enzymology , Protein Kinases/biosynthesis , Protein Serine-Threonine Kinases , Protein-Tyrosine Kinases/biosynthesis , RNA, Messenger/biosynthesis , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Chlorocebus aethiops , Cloning, Molecular , Exons , Humans , Mice , Molecular Sequence Data , PC12 Cells , Polymerase Chain Reaction , Rats , Recombinant Fusion Proteins/biosynthesis , Sequence Homology, Amino Acid , Transcription, Genetic , TransfectionABSTRACT
A novel protein kinase, the Esk kinase, has been isolated from an embryonal carcinoma (EC) cell line by using an expression cloning strategy. Sequence analysis of two independent cDNA clones (2.97 and 2.85 kb) suggested the presence of two Esk isoforms in EC cells. The esk-1 cDNA sequence predicted an 857-amino-acid protein kinase with a putative membrane-spanning domain, while the esk-2 cDNA predicted an 831-amino-acid kinase which lacked this domain. In adult mouse cells, esk mRNA levels were highest in tissues possessing a high proliferation rate or a sizeable stem cell compartment, suggesting that the Esk kinase may play some role in the control of cell proliferation or differentiation. As anticipated from the screening procedure, bacterial expression of the Esk kinase reacted with antiphosphotyrosine antibodies on immunoblots. Furthermore, in in vitro kinase assays, the Esk kinase was shown to phosphorylate both itself and the exogenous substrate myelin basic protein on serine, threonine, and tyrosine residues, confirming that the Esk kinase is a novel member of the serine/threonine/tyrosine family of protein kinases.
Subject(s)
Protein Kinases/genetics , Amino Acid Sequence , Animals , Base Sequence , Cell Compartmentation , Cell Membrane/enzymology , Cloning, Molecular , Cytoplasm/enzymology , DNA/genetics , Gene Expression , In Vitro Techniques , Mice , Molecular Sequence Data , Phosphoproteins/genetics , Phosphoserine/metabolism , Phosphothreonine/metabolism , Phosphotyrosine , Protein Kinases/metabolism , RNA, Messenger/genetics , Sequence Alignment , Substrate Specificity , Tumor Cells, Cultured , Tyrosine/analogs & derivatives , Tyrosine/metabolismABSTRACT
We have cloned a novel kinase (STY) from an embryonal carcinoma cell line. Sequence analysis of the STY cDNA reveals that it shares sequence homology with serine/threonine-type kinases and yet the bacterial expression product of the STY cDNA appears to have serine-, threonine-, and tyrosine-phosphorylating activities. The predicted STY protein is highly basic and contains a putative nuclear localization signal. During differentiation, two new mRNAs were detected in addition to the embryonic transcript.
