title: Differential signaling during macropinocytosis in response to M-CSF and PMA in macrophages.
abstract: The cellular movements that construct a macropinosome have a corresponding sequence of chemical transitions in the cup-shaped region of plasma membrane that becomes the macropinosome. To determine the relative positions of type I phosphatidylinositol 3-kinase (PI3K) and phospholipase C (PLC) in this pathway, we analyzed macropinocytosis in macrophages stimulated by the growth factor macrophage-colony-stimulating factor (M-CSF) and by the diacylglycerol (DAG) analog phorbol 12-myristate 13-acetate (PMA). In cells stimulated with M-CSF, microscopic imaging of fluorescent probes for intracellular lipids indicated that the PI3K product phosphatidylinositol (3,4,5)-trisphosphate (PIP3) appeared in cups just prior to DAG. We then tested the hypothesis that PMA and DAG function after PI3K and prior to Ras and protein kinase C (PKC) during macropinosome formation in macrophages. Although the PI3K target Akt was activated by M-CSF, the Akt inhibitor MK-2206 did not inhibit macropinocytosis. The phospholipase C (PLC) inhibitor U73122 blocked macropinocytosis by M-CSF but not PMA. Macropinocytosis in response to M-CSF and PMA was inhibited by the Ras inhibitor farnesyl thiosalicylate (FTS), by the PKC inhibitor Calphostin C and by the broad specificity inhibitor rottlerin. These studies support a model in which M-CSF stimulates PI3K in macropinocytic cups, and the resulting increase in PIP3 activates PLC, which in turn generates DAG necessary for activation of PKC, Ras and the late stages of macropinosome closure.
authors: Department of Microbiology and Immunology, University of Michigan Medical School Ann Arbor, MI, USA.
authors: Sei
authors: S
authors: Yoshida
authors: Department of Microbiology and Immunology, University of Michigan Medical School Ann Arbor, MI, USA.
authors: Isabella
authors: I
authors: Gaeta
authors: Department of Microbiology and Immunology, University of Michigan Medical School Ann Arbor, MI, USA.
authors: Regina
authors: R
authors: Pacitto
authors: Department of Microbiology and Immunology, University of Michigan Medical School Ann Arbor, MI, USA.
authors: Lydia
authors: L
authors: Krienke
authors: Department of Microbiology and Immunology, University of Michigan Medical School Ann Arbor, MI, USA.
authors: Olivia
authors: O
authors: Alge
authors: Department of Microbiology and Immunology, University of Michigan Medical School Ann Arbor, MI, USA.
authors: Brian
authors: B
authors: Gregorka
authors: Department of Microbiology and Immunology, University of Michigan Medical School Ann Arbor, MI, USA.
authors: Joel A
authors: JA
authors: Swanson
keywords: Ras; diacylglycerol; macropinocytosis; phosphatidylinositol (3,4,5)-trisphosphate; phospholipase C; protein kinase C