Assuntos
Fosfoproteínas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Células COS , Chlorocebus aethiops , Clonagem Molecular/métodos , Primers do DNA , DNA Complementar , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Biblioteca Gênica , Genes Reporter , Microscopia de Fluorescência , Dados de Sequência Molecular , Fosfoproteínas/análise , Fosfoproteínas/genética , Fosforilação , Plasmídeos , Proteínas Tirosina Quinases/genética , Proteínas Recombinantes de Fusão/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Especificidade por SubstratoRESUMO
Immunoreceptors such as the high affinity IgE receptor, FcepsilonRI, and T-cell receptor-associated proteins share a common motif, the immunoreceptor tyrosine-based activation motif (ITAM). We used the yeast tribrid system to identify downstream effectors of the phosphorylated FcepsilonRI ITAM-containing subunits beta and gamma. One novel cDNA was isolated that encodes a protein that is phosphorylated on tyrosine, contains a Src-homology 2 (SH2) domain, inositolpolyphosphate 5-phosphatase activity, three NXXY motifs, several proline-rich regions, and is called SHIP. Mutation of the conserved tyrosine or leucine residues within the FcepsilonRI beta or gamma ITAMs eliminates SHIP binding and indicates that the SHIP-ITAM interaction is specific. SHIP also binds to ITAMs from the CD3 complex and T cell receptor zeta chain in vitro. SHIP protein possesses both phosphatidylinositol-3,4,5-trisphosphate 5'-phosphatase and inositol-1,3,4,5-tetrakisphosphate 5'-phosphatase activity. Phosphorylation of SHIP by a protein-tyrosine kinase, Lck, results in a reduction in enzyme activity. FcepsilonRI activation induces the association of several tyrosine phosphoproteins with SHIP. SHIP is constitutively tyrosine-phosphorylated and associated with Shc and Grb2. These data suggest that SHIP may serve as a multifunctional linker protein in receptor activation.
Assuntos
Monoéster Fosfórico Hidrolases/metabolismo , Receptores de IgE/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , DNA Complementar , Glutationa Transferase/genética , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Monoéster Fosfórico Hidrolases/genética , Fosforilação , Ligação Proteica , RNA Mensageiro/genética , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Tirosina/metabolismo , Domínios de Homologia de srcRESUMO
Mitogen-activated protein kinase (MAPK) is activated in many cell types in response to growth factors during the G0-G1 transition in the cell cycle. We investigated the effects of platelet-derived growth factor (PDGF) AA and PDGF BB on activation of MAPK in human dermal fibroblasts, and asked whether its activation correlates with proliferative responses of human fibroblasts to PDGF AA and PDGF BB. Treatment with either PDGF isoform for 20 min resulted in equal phosphorylation of MAPK as visualized by gel shifts in Western blotting with anti-MAPK polyclonal antibody. This finding was confirmed by measurements of MAPK activity in response to increasing doses (2-20 ng/ml) of PDGF AA and PDGF BB in in vitro assays with myelin basic protein as a substrate. PDGF AA was slightly less potent than PDGF BB, but both growth factors induced similar maximal activations. Kinetics of activation were also similar for both isoforms, with maximal induction of MAPK at 10-20 min after growth factor addition followed by a gradual decline to control levels at 1 h. Activation of MAPK by both PDGF isoforms was also confirmed by measuring myelin basis protein phosphorylation in MAPK immunoprecipitates. Thus both PDGF AA and PDGF BB are potent activators of MAPK in human dermal fibroblasts. In contrast, PDGF BB elicited a strong mitogenic response, while PDGF AA had no significant effect on DNA synthesis in human dermal fibroblasts. These data indicate that acute activation of MAPK is not sufficient to stimulate cells to progress through cell cycle. PDGF AA may have other biologic functions or may play a co-stimulatory role in proliferation of human dermal fibroblasts.