A novel mechanism of ERK1/2 regulation in smooth muscle involving acetylation of the ERK1/2 scaffold IQGAP1.

Ceramide, a bioactive lipid and signaling molecule associated with cardiovascular disease, is known to activate extracellular signal regulated kinases 1 and 2 (ERK1/2). Here, we determined that the effect of ceramide on ERK1/2 is mediated by ceramide signaling on an ERK scaffold protein, IQ motif containing GTPase activating protein 1 (IQGAP1). Experiments were performed with aortic smooth muscle cells using inhibitor screening, small interfering RNA (siRNA), immunoprecipitation (IP), immunoblots and bioinformatics.

Hierarchical scaffolding of an ERK1/2 activation pathway.

Background: Scaffold proteins modulate cellular signaling by facilitating assembly of specific signaling pathways. However, there is at present little information if and how scaffold proteins functionally interact with each other.

Results: Here, we show that two scaffold proteins, caveolin-1 and IQGAP1, are required for phosphorylation of the actin associated pool of extracellular signal regulated kinase 1 and 2 (ERK1/2) in response to protein kinase C activation.

Vasoconstrictor-induced endocytic recycling regulates focal adhesion protein localization and function in vascular smooth muscle.

Turnover of focal adhesions (FAs) is known to be critical for cell migration and adhesion of proliferative vascular smooth muscle (VSM) cells. However, it is often assumed that FAs in nonmigratory, differentiated VSM (dVSM) cells embedded in the wall of healthy blood vessels are stable structures. Recent work has demonstrated agonist-induced actin polymerization and Src-dependent FA phosphorylation in dVSM cells, suggesting that agonist-induced FA remodeling occurs.

The focal adhesion: a regulated component of aortic stiffness.

Increased aortic stiffness is an acknowledged predictor and cause of cardiovascular disease. The sources and mechanisms of vascular stiffness are not well understood, although the extracellular matrix (ECM) has been assumed to be a major component. We tested here the hypothesis that the focal adhesions (FAs) connecting the cortical cytoskeleton of vascular smooth muscle cells (VSMCs) to the matrix in the aortic wall are a component of aortic stiffness and that this component is dynamically regulated.

Stimulus-specific activation and actin dependency of distinct, spatially separated ERK1/2 fractions in A7r5 smooth muscle cells.

A proliferative response of smooth muscle cells to activation of extracellular signal regulated kinases 1 and 2 (ERK1/2) has been linked to cardiovascular disease. In fully differentiated smooth muscle, however, ERK1/2 activation can also regulate contraction. Here, we use A7r5 smooth muscle cells, stimulated with 12-deoxyphorbol 13-isobutylate 20-acetate (DPBA) to induce cytoskeletal remodeling or fetal calf serum (FCS) to induce proliferation, to identify factors that determine the outcomes of ERK1/2 activation in smooth muscle.

Src modulates contractile vascular smooth muscle function via regulation of focal adhesions.

Src is a known regulator of focal adhesion turnover in migrating cells; but, in contrast, Src is generally assumed to play little role in differentiated, contractile vascular smooth muscle (dVSM). The goal of the present study was to determine if Src-family kinases regulate focal adhesion proteins and how this might affect contractility of non-proliferative vascular smooth muscle. We demonstrate here, through the use of phosphotyrosine screening, deconvolution microscopy imaging, and differential centrifugation, that the activity of Src family kinases in aorta is regulated by the alpha agonist and vasoconstrictor phenylephrine, and leads to focal adhesion protein phosphorylation and remodeling in dVSM.

h3/Acidic calponin: an actin-binding protein that controls extracellular signal-regulated kinase 1/2 activity in nonmuscle cells.

Migration of fibroblasts is important in wound healing. Here, we demonstrate a role and a mechanism for h3/acidic calponin (aCaP, CNN3) in REF52.2 cell motility, a fibroblast line rich in actin filaments.

Par-4: a new activator of myosin phosphatase.

Myosin phosphatase (MP) is a key regulator of myosin light chain (LC20) phosphorylation, a process essential for motility, apoptosis, and smooth muscle contractility. Although MP inhibition is well studied, little is known about MP activation. We have recently demonstrated that prostate apoptosis response (Par)-4 modulates vascular smooth muscle contractility.

Par-4 is an essential downstream target of DAP-like kinase (Dlk) in Dlk/Par-4-mediated apoptosis.

Prostate apoptosis response-4 (Par-4) was initially identified as a gene product up-regulated in prostate cancer cells undergoing apoptosis. In rat fibroblasts, coexpression of Par-4 and its interaction partner DAP-like kinase (Dlk, which is also known as zipper-interacting protein kinase [ZIPK]) induces relocation of the kinase from the nucleus to the actin filament system, followed by extensive myosin light chain (MLC) phosphorylation and induction of apoptosis. Our analyses show that the synergistic proapoptotic effect of Dlk/Par-4 complexes is abrogated when either Dlk/Par-4 interaction or Dlk kinase activity is impaired.

The pro-apoptotic protein Par-4 facilitates vascular contractility by cytoskeletal targeting of ZIPK.

Par-4 (prostate apoptosis response 4) is a pro-apoptotic protein and tumour suppressor that was originally identified as a gene product up-regulated during apoptosis in prostate cancer cells. Here, we show, for the first time, that Par-4 is expressed and co-localizes with the actin filament bundles in vascular smooth muscle. Furthermore, we demonstrate that targeting of ZIPK to the actin filaments, as observed upon PGF-2alpha stimulation, is inhibited by the presence of a cell permeant Par-4 decoy peptide.

Par-4-mediated recruitment of Amida to the actin cytoskeleton leads to the induction of apoptosis.

Par-4 (prostate apoptosis response-4) sensitizes cells to apoptotic stimuli, but the exact mechanisms are still poorly understood. Using Par-4 as bait in a yeast two-hybrid screen, we identified Amida as a novel interaction partner, a ubiquitously expressed protein which has been suggested to be involved in apoptotic processes. Complex formation of Par-4 and Amida occurs in vitro and in vivo and is mediated via the C-termini of both proteins, involving the leucine zipper of Par-4.

Binding of Par-4 to the actin cytoskeleton is essential for Par-4/Dlk-mediated apoptosis.

Prostate apoptosis response-4 (Par-4) is a 38-kDa protein originally identified as a gene product upregulated in prostate cancer cells undergoing apoptosis. Cell death mediated by Par-4 and its interaction partner DAP like kinase (Dlk) is characterized by dramatic changes of the cytoskeleton. To uncover the role of the cytoskeleton in Par-4/Dlk-mediated apoptosis, we analyzed Par-4 for a direct association with cytoskeletal structures.

Ectopic expression of Par-4 leads to induction of apoptosis in CNS tumor cell lines.

Prostate apoptosis response-4 (Par-4) is a pro-apoptotic protein originally identified as a gene product upregulated in prostate tumor cells undergoing apoptosis. Down-regulation of Par-4 has been linked to several cancers. Since Par-4 also plays a crucial role in neuronal apoptosis, we investigated the expression of Par-4 in tumor cell lines derived from representative tumor types of the CNS, including primitive neuroectodermal tumor (PNET), medulloblastoma, neuroblastoma and glioma of human, rat and murine origin.

The rat homologue of Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP) associates with actin filaments, recruits N-WASP from the nucleus, and mediates mobilization of actin from stress fibers in favor of filopodia formation.

We cloned and characterized the rat homologue of the Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP). Rat WIP shows 86% amino acid sequence identity to human WIP. Northern analyses revealed two major mRNA species of 5.