ABSTRACT
SHIP-2 is a phosphoinositidylinositol 3,4,5 trisphosphate (PtdIns[3,4,5]P3) 5-phosphatase that contains an NH2-terminal SH2 domain, a central 5-phosphatase domain, and a COOH-terminal proline-rich domain. SHIP-2 negatively regulates insulin signaling. In unstimulated cells, SHIP-2 localized in a perinuclear cytosolic distribution and at the leading edge of the cell. Endogenous and recombinant SHIP-2 localized to membrane ruffles, which were mediated by the COOH-terminal proline-rich domain. To identify proteins that bind to the SHIP-2 proline-rich domain, yeast two-hybrid screening was performed, which isolated actin-binding protein filamin C. In addition, both filamin A and B specifically interacted with SHIP-2 in this assay. SHIP-2 coimmunoprecipitated with filamin from COS-7 cells, and association between these species did not change after epidermal growth factor stimulation. SHIP-2 colocalized with filamin at Z-lines and the sarcolemma in striated muscle sections and at membrane ruffles in COS-7 cells, although the membrane ruffling response was reduced in cells overexpressing SHIP-2. SHIP-2 membrane ruffle localization was dependent on filamin binding, as SHIP-2 was expressed exclusively in the cytosol of filamin-deficient cells. Recombinant SHIP-2 regulated PtdIns(3,4,5)P3 levels and submembraneous actin at membrane ruffles after growth factor stimulation, dependent on SHIP-2 catalytic activity. Collectively these studies demonstrate that filamin-dependent SHIP-2 localization critically regulates phosphatidylinositol 3 kinase signaling to the actin cytoskeleton.
Subject(s)
Actins/metabolism , Contractile Proteins/metabolism , Microfilament Proteins/metabolism , Phosphoric Monoester Hydrolases/metabolism , Amino Acid Sequence , Animals , COS Cells/drug effects , COS Cells/enzymology , Cell Membrane/enzymology , Contractile Proteins/genetics , Epidermal Growth Factor/pharmacology , Filamins , Humans , Inositol Polyphosphate 5-Phosphatases , Melanoma , Mice , Microfilament Proteins/genetics , Molecular Sequence Data , Muscle Fibers, Skeletal/chemistry , Muscle, Skeletal/chemistry , Muscle, Skeletal/cytology , Myocardium/chemistry , Myocardium/cytology , Oligonucleotide Probes , Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases , Phosphatidylinositols/metabolism , Phosphoric Monoester Hydrolases/analysis , Phosphoric Monoester Hydrolases/genetics , Tumor Cells, Cultured , Two-Hybrid System Techniques , YeastsABSTRACT
To further understand the relative roles of the pituitary gland and ACTH in the regulation of mRNAs encoding proteins that are essential for adrenal development, we investigated the effects of, first, an ACTH infusion and labour in intact fetuses and, secondly, the effect of an ACTH infusion to fetuses with and without a pituitary gland, on the relative abundance of the mRNA encoding for the ACTH receptor (MC2R), steroidogenic factor 1 (SF-1), cholesterol side-chain cleavage enzyme (P450(scc)), 3beta-hydroxysteroid dehydrogenase (3betaHSD) and 17alpha-hydroxylase (P450(C17)) in the fetal adrenal gland. ACTH(1-24) infusion (14.7 pmol/kg per h) to intact fetuses was without effect on the abundance of mRNA encoding MC2R and SF-1, irrespective of whether the infusion was given for 18 (115-132 days of gestation) or 32 days (115 days to term (147 days of gestation)). Hypophysectomy (HX) did not alter the expression of MC2R mRNA; however, the abundance of SF-1 mRNA fell by approximately 50% following the removal of the pituitary gland. ACTH(1-24) infusion to HX fetuses failed to restore levels of SF-1 mRNA to that seen in intact animals. P450(scc) and 3betaHSD mRNAs were increased by ACTH(1-24) infusion for 18 days in intact animals, although no effects of the infusion were seen on P450(C17) mRNA levels. For all three of these mRNAs, there was a significant increase in their abundance between 132 days of gestation and term in intact fetuses. By term, ACTH(1-24) infusion was without any additional effect on their abundance. HX decreased the expression of P450(scc), 3betaHSD and P450(C17) mRNAs, while ACTH(1-24) infusion to HX fetuses increased the expression of these mRNAs to levels seen in intact animals. There were significant correlations between the abundance of the mRNA for P450(scc), 3betaHSD and P450(C17), but not MC2R and SF-1, and premortem plasma cortisol concentrations. These results emphasise the importance of the pituitary gland and ACTH in the regulation of the enzymes involved in adrenal steroidogenesis. Factors in addition to ACTH may also play some role, as the infusion was not always effective in increasing the abundance of the mRNAs. Surprisingly, the mRNA for MC2R and SF-1 did not appear to be regulated by ACTH in the late-gestation ovine fetus, though a pituitary-dependent factor may be involved in the regulation of SF-1 mRNA abundance.
Subject(s)
Adrenal Glands/embryology , Adrenocorticotropic Hormone/physiology , Gene Expression Regulation, Developmental/physiology , Pituitary Gland/physiology , Sheep/embryology , Adrenal Glands/enzymology , Adrenal Glands/physiology , Adrenocorticotropic Hormone/pharmacology , Animals , Embryonic and Fetal Development/physiology , Enzymes/genetics , Female , Gene Expression Regulation, Developmental/drug effects , Hydrocortisone/blood , Labor, Obstetric/physiology , Pregnancy , RNA, Messenger/genetics , Sheep/physiologyABSTRACT
LIM domains are double zinc-finger motifs that mediate protein interactions between transcription factors, cytoskeletal and signaling proteins. This review outlines the protein interactions mediated by LIM domains with signaling proteins including tyrosine and serine/threonine kinases and phosphatases.
