Adam D. Munday

The SH2-containing inositol polyphosphate 5-phosphatase, SHIP-2, binds filamin and regulates submembraneous actin.

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.

Increased levels of phosphatidylinositol 3-kinase activity in colorectal tumors†

Phosphatidylinositol 3‐kinase (PI 3‐kinase), an enzyme that phosphorylates inositol phospholipids at the D‐3 position of the inositol ring, has been implicated in the signaling pathways regulating cell growth by virtue of its activation in response to various mitogenic stimuli. In spite of the considerable attention PI 3‐kinase has received with regard to its possible role in the mitogenic pathways in hematopoietic malignancies, there are few reports of investigations into PI 3‐kinase activity in solid tumors.

Identification of a Novel Domain in Two Mammalian Inositol-polyphosphate 5-Phosphatases That Mediates Membrane Ruffle Localization THE INOSITOL 5-PHOSPHATASE SKIP LOCALIZES TO THE ENDOPLASMIC RETICULUM AND TRANSLOCATES TO MEMBRANE RUFFLES FOLLOWING EPIDERMAL GROWTH FACTOR STIMULATION

SKIP (skeletal muscle andkidney enriched inositolphosphatase) is a recently identified phosphatidylinositol 3,4,5-trisphosphate- and phosphatidylinositol 4,5-bisphosphate-specific 5-phosphatase. In this study, we investigated the intracellular localization of SKIP. Indirect immunofluorescence and subcellular fractionation showed that, in serum-starved cells, both endogenous and recombinant SKIP colocalized with markers of the endoplasmic reticulum (ER). Following epidermal growth factor (EGF) stimulation, SKIP transiently translocated to plasma membrane ruffles and colocalized with submembranous actin. Data base searching demonstrated a novel 128-amino acid domain in the C terminus of SKIP, designated SKICH for SKIP carboxyl homology, which is also found in the 107-kDa 5-phosphatase PIPP and in members of the TRAF6-binding protein family. Recombinant SKIP lacking the SKICH domain localized to the ER, but did not translocate to membrane ruffles following EGF stimulation. The SKIP SKICH domain showed perinuclear localization and mediated EGF-stimulated plasma membrane ruffle localization. The SKICH domain of the 5-phosphatase PIPP also mediated plasma membrane ruffle localization. Mutational analysis identified the core sequence within the SKICH domain that mediated constitutive membrane association and C-terminal sequences unique to SKIP that contributed to ER localization. Collectively, these studies demonstrate a novel membrane-targeting domain that serves to recruit SKIP and PIPP to membrane ruffles.

Mitochondria are the functional intracellular target for a photosensitizing boronated porphyrin

A photosensitizing boron-containing porphyrin derivative denoted BOPP, which is selectively localised into mitochondria, has been tested on Namalwa cells, in each of two genetic configurations: rho+ cells containing normal mtDNA and mitochondrial respiratory functions, or rho0 cells lacking mtDNA and devoid of mitochondrial oxidative phosphorylation. After short-term cellular uptake for 18 h, BOPP (30 micrograms/ml) was not cytotoxic, but did show marked phototoxicity in Namalwa rho+ cells, concomitant with substantial reduction of mitochondrial respiratory activity. After long-term (3 days or more) exposure to BOPP without light, growth of Namalwa rho+ cells was inhibited at concentrations significantly above 30 micrograms/ml. At such concentrations BOPP was shown to have direct inhibitory effects on mitochondrial azide-sensitive respiration of p+ cells. By contrast, BOPP showed neither cytotoxic nor phototoxic effects in rho0 cells. These results indicate functional mitochondria to be a major cellular target in vivo after BOPP uptake and photoactivation.

Leukocyte integrin Mac-1 regulates thrombosis via interaction with platelet GPIbα

Inflammation and thrombosis occur together in many diseases. The leukocyte integrin Mac-1 (also known as integrin αMβ2, or CD11b/CD18) is crucial for leukocyte recruitment to the endothelium, and Mac-1 engagement of platelet GPIbα is required for injury responses in diverse disease models. However, the role of Mac-1 in thrombosis is undefined. Here we report that mice with Mac-1 deficiency (Mac-1−/−) or mutation of the Mac-1-binding site for GPIbα have delayed thrombosis after carotid artery and cremaster microvascular injury without affecting parameters of haemostasis. Adoptive wild-type leukocyte transfer rescues the thrombosis defect in Mac-1−/− mice, and Mac-1-dependent regulation of the transcription factor Foxp1 contributes to thrombosis as evidenced by delayed thrombosis in mice with monocyte-/macrophage-specific overexpression of Foxp1. Antibody and small-molecule targeting of Mac-1:GPIbα inhibits thrombosis. Our data identify a new pathway of thrombosis involving leukocyte Mac-1 and platelet GPIbα, and suggest that targeting this interaction has anti-thrombotic therapeutic potential with reduced bleeding risk.

Corrigendum: Leukocyte integrin Mac-1 regulates thrombosis via interaction with platelet GPIbα

This corrects the article DOI: 10.1038/ncomms15559.

I’m TORC1-ing platelets and thromboembolism

In this issue of Blood, Yang et al investigate the mechanism behind the aging-related increase in venous thrombosis and provide evidence for the novel role of mammalian target of rapamycin complex 1 (mTORC1) in mediating the observed increase in mean platelet volume, a predictor of unprovoked venous thromboembolism.1-3

Mechanical Regulation of Von Willebrand Factor A1 Domain

Regulation of the bond between platelet Glycoprotein Ib (GPIb) and the von Willebrand Factor (VWF) A1 domain is critical to the balance between hemostasis and thrombosis, particularly in high shear conditions. The GPIbα-A1 interaction is known to be activated by shear stress and/or tensile mechanical force, but the structural basis of this mechanical regulation remained unknown. To address this question, we expressed a number of A1 constructs that differed in one or more residues or domains and anchored them to the surface at known concentrations and orientations. We then measured rolling velocities of platelets or GPIb-coated microspheres to determine the importance of different structural regions and the manner in which force is applied. We show that an intrinsically disordered peptide region N-terminal to the A1 domain has strong inhibitory activity. In addition, the A1-GPIb bond is much stronger when the A1 domain is anchored via the C-terminus rather than the N-terminus. This suggests that shear stress activates A1-GPIb binding in two ways. First, it applies tensile force across multimeric VWF, activating the A1 domain by dislodging this inhibitory region. Second, it applies tensile force across the A1-GPIb bond, inducing a conformational change in the C-terminal region of VWF that allosterically activates the A1 domain, perhaps in a way analogous to that seen in integrins.