Functional interaction between the epidermal growth factor receptor and c-Src kinase activity

AbstractTo study the relationship between the tyrosine kinase c-Src and the epidermal growth factor receptor (EGF-R), we used the breast cancer cell line ZR75-1, which was transfected with the EGF-R. The EGF-R transfected cell line expressed 60 times more EGF-R than a control cell line transfected with the empty vector. In the presence of EGF, the EGF-R over-expressing cell line grew much faster than the control cell line. Both cell lines expressed approximately equal amounts of c-Src. However, the cell line over-expressing the EGF-R showed a twofold enhancement of c-Src kinase activity after EGF stimulation. The activation of c-Src kinase by EGF was confirmed in other EGF-R expressing cell types

β-1,3-Glucan-Induced Host Phospholipase D Activation Is Involved in Aspergillus fumigatus Internalization into Type II Human Pneumocyte A549 Cells

The internalization of Aspergillus fumigatus into lung epithelial cells is a process that depends on host cell actin dynamics. The host membrane phosphatidylcholine cleavage driven by phospholipase D (PLD) is closely related to cellular actin dynamics. However, little is known about the impact of PLD on A. fumigatus internalization into lung epithelial cells. Here, we report that once germinated, A. fumigatus conidia were able to stimulate host PLD activity and internalize more efficiently in A549 cells without altering PLD expression. The internalization of A. fumigatus in A549 cells was suppressed by the downregulation of host cell PLD using chemical inhibitors or siRNA interference. The heat-killed swollen conidia, but not the resting conidia, were able to activate host PLD. Further, β-1,3-glucan, the core component of the conidial cell wall, stimulated host PLD activity. This PLD activation and conidia internalization were inhibited by anti-dectin-1 antibody. Indeed, dectin-1, a β-1,3-glucan receptor, was expressed in A549 cells, and its expression profile was not altered by conidial stimulation. Finally, host cell PLD1 and PLD2 accompanied A. fumigatus conidia during internalization. Our data indicate that host cell PLD activity induced by β-1,3-glucan on the surface of germinated conidia is important for the efficient internalization of A. fumigatus into A549 lung epithelial cells

Phospholipase D signaling: orchestration by PIP2 and small GTPases

Hydrolysis of phosphatidylcholine by phospholipase D (PLD) leads to the generation of the versatile lipid second messenger, phosphatidic acid (PA), which is involved in fundamental cellular processes, including membrane trafficking, actin cytoskeleton remodeling, cell proliferation and cell survival. PLD activity can be dramatically stimulated by a large number of cell surface receptors and is elaborately regulated by intracellular factors, including protein kinase C isoforms, small GTPases of the ARF, Rho and Ras families and, particularly, by the phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 is well known as substrate for the generation of second messengers by phospholipase C, but is now also understood to recruit and/or activate a variety of actin regulatory proteins, ion channels and other signaling proteins, including PLD, by direct interaction. The synthesis of PIP2 by phosphoinositide 5-kinase (PIP5K) isoforms is tightly regulated by small GTPases and, interestingly, by PA as well, and the concerted formation of PIP2 and PA has been shown to mediate receptor-regulated cellular events. This review highlights the regulation of PLD by membrane receptors, and describes how the close encounter of PLD and PIP5K isoforms with small GTPases permits the execution of specific cellular functions

Direct stimulation of receptor-controlled phospholipase D1 by phospho-cofilin

The activity state of cofilin, which controls actin dynamics, is driven by a phosphorylation–dephosphorylation cycle. Phosphorylation of cofilin by LIM-kinases results in its inactivation, a process supported by 14-3-3ζ and reversed by dephosphorylation by slingshot phosphatases. Here we report on a novel cellular function for the phosphorylation–dephosphorylation cycle of cofilin. We demonstrate that muscarinic receptor-mediated stimulation of phospholipase D1 (PLD1) is controlled by LIM-kinase, slingshot phosphatase as well as 14-3-3ζ, and requires phosphorylatable cofilin. Cofilin directly and specifically interacts with PLD1 and upon phosphorylation by LIM-kinase1, stimulates PLD1 activity, an effect mimicked by phosphorylation-mimic cofilin mutants. The interaction of cofilin with PLD1 is under receptor control and encompasses a PLD1-specific fragment (aa 585–712). Expression of this fragment suppresses receptor-induced cofilin–PLD1 interaction as well as PLD stimulation and actin stress fiber formation. These data indicate that till now designated inactive phospho-cofilin exhibits an active cellular function, and suggest that phospho-cofilin by its stimulatory effect on PLD1 may control a large variety of cellular functions

The Dual Effect of Rac2 on Phospholipase D2 Regulation That Explains both the Onset and Termination of Chemotaxis ▿

We document a biphasic effect of Rac2 on the activation and inhibition of PLD2. Cells overexpressing Rac2 and PLD2 simultaneously show a robust initial (<10 min) response toward a chemoattractant that is later (>30 min) greatly diminished over PLD2-only controls. The first phase is due to the presence of a Rac2-PLD2 positive-feedback loop. To explain the mechanism for the Rac2-led PLD2 inhibition (the second phase), we used leukocytes from wild-type (WT) and Rac2−/− knockout mice. Rac2−/− cells displayed an enhanced PLD2 (but not PLD1) enzymatic activity, confirming the inhibitory role of Rac2. Late inhibitory responses on PLD2 due to Rac2 were reversed in the presence of phosphatidylinositol 4,5-bisphosphate (PIP2) both in vitro (purified GST-PH-PLD2, where GST is glutathione S-transferase and PH is pleckstrin homology) and in vivo. Coimmunoprecipitation and immunofluorescence microscopy indicated that PLD2 and Rac2 remain together. The presence of an “arc” of Rac2 at the leading edge of leukocyte pseudopodia and PLD2 physically posterior to this wave of Rac2 was observed in late chemotaxis. We propose Rac-led inhibition of PLD2 function is due to sterical interference of Rac with PLD2's PH binding site to the membrane and deprivation of the PIP2. This work supports the importance of functional interactions between PLD and Rac in the biological response of cell migration

DLC1 Activation Requires Lipid Interaction through a Polybasic Region Preceding the RhoGAP Domain

Deleted in Liver Cancer 1 (DLC1) is a GTPase-activating protein (GAP) with specificity for RhoA, RhoB, and RhoC that is frequently deleted in various tumor types. By inactivating these small GTPases, DLC1 controls actin cytoskeletal remodeling and biological processes such as cell migration and proliferation. Here we provide evidence that DLC1 binds to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) through a previously unrecognized polybasic region (PBR) adjacent to its RhoGAP domain. Importantly, PI(4,5)P2-containing membranes are shown to stimulate DLC1 GAP activity in vitro. In living cells, a DLC1 mutant lacking an intact PBR inactivated Rho signaling less efficiently and was severely compromised in suppressing cell spreading, directed migration, and proliferation. We therefore propose that PI(4,5)P2 is an important cofactor in DLC1 regulation in vivo and that the PBR is essential for the cellular functions of the protein