ABSTRACT
The proliferation and differentiation of cells of many lineages are regulated by secreted proteins known as cytokines. Cytokines exert their biological effect through binding to cell-surface receptors that are associated with one or more members of the JAK family of cytoplasmic tyrosine kinases. Cytokine-induced receptor dimerization leads to the activation of JAKs, rapid tyrosine-phosphorylation of the cytoplasmic domains, and subsequent recruitment of various signalling proteins, including members of the STAT family of transcription factors, to the receptor complex. Using the yeast two-hybrid system, we have now isolated a new SH2-domain-containing protein, JAB, which is a JAK-binding protein that interacts with the Jak2 tyrosine-kinase JH1 domain. JAB is structurally related to CIS, a cytokine-inducible SH2 protein. Interaction of JAB with Jak1, Jak2 or Jak3 markedly reduces their tyrosine-kinase activity and suppresses the tyrosine-phosphorylation and activation of STATs. JAB and CIS appear to function as negative regulators in the JAK signalling pathway.
Subject(s)
Carrier Proteins/analysis , Enzyme Inhibitors/analysis , Intracellular Signaling Peptides and Proteins , Milk Proteins , Protein-Tyrosine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins , Repressor Proteins , Signal Transduction , src Homology Domains , Amino Acid Sequence , Animals , Carrier Proteins/chemistry , Carrier Proteins/genetics , Carrier Proteins/physiology , Cell Line , Cloning, Molecular , DNA, Complementary , DNA-Binding Proteins/genetics , Erythropoietin/antagonists & inhibitors , Erythropoietin/physiology , Gene Expression Regulation , Genes, Reporter , Humans , Immediate-Early Proteins/chemistry , Immediate-Early Proteins/physiology , Interleukin-2/antagonists & inhibitors , Interleukin-2/physiology , Interleukin-6/antagonists & inhibitors , Interleukin-6/physiology , Janus Kinase 2 , Mice , Molecular Sequence Data , Phosphorylation , Proto-Oncogene Proteins c-fos/genetics , Rats , Recombinant Fusion Proteins/genetics , STAT3 Transcription Factor , STAT5 Transcription Factor , Saccharomyces cerevisiae , Suppressor of Cytokine Signaling 1 Protein , Suppressor of Cytokine Signaling Proteins , Trans-Activators/geneticsABSTRACT
A mammalian plasma membrane protein(s) which catalyzes ATP-dependent transbilayer movement (flip-flop) of phosphatidylserine (PS) has been suggested to be involved in the formation and maintenance of membrane lipid asymmetry. Flip-flop of PS in the cell surface of nucleated cells was first described by O. C. Martin and R. E. Pagano (1987, J. Biol. Chem. 262, 5890-5898). It has been suggested that flip-flop is involved in the internalization of exogenous PS in cultured cells. In the present study we report that incubation with an excess amount of PS is cytotoxic to Chinese hamster ovary (CHO) cells, while the same amount of phosphatidylcholine gives no effect. This effect allowed us to obtain PS-resistant cells among mutagenized CHO cells. Endocytosis-independent internalization of exogenous fluorescent PS analog was defective in 40% of the PS-resistant mutants. One of the mutants, PSR (phosphatidylserine resistant) 406 was further characterized. Unlike wild-type CHO cells, this mutant did not transport fluorescent PS significantly at 15 degrees C. Fluorescent PS was not metabolized at 15 degrees C in either wild-type or mutant cells. These results suggest that transbilayer movement of cell surface PS is defective in PS-resistant cells.
