[Show abstract][Hide abstract] ABSTRACT: Activation of the small GTPase RhoA following angiotensin II stimulation is known to result in actin reorganization and stress
fiber formation. Full activation of RhoA, by angiotensin II, depends on the scaffolding protein β-arrestin 1, although the
mechanism behind its involvement remains elusive. Here we uncover a novel partner and function for β-arrestin 1, namely, in
binding to ARHGAP21 (also known as ARHGAP10), a known effector of RhoA activity, whose GTPase-activating protein (GAP) function
it inhibits. Using yeast two-hybrid screening, a peptide array, in vitro binding studies, truncation analyses, and coimmunoprecipitation techniques, we show that β-arrestin 1 binds directly to ARHGAP21
in a region that transects the RhoA effector GAP domain. Moreover, we show that the level of a complex containing β-arrestin
1 and ARHGAP21 is dynamically increased following angiotensin stimulation and that the kinetics of this interaction modulates
the temporal activation of RhoA. Using information gleaned from a peptide array, we developed a cell-permeant peptide that
serves to inhibit the interaction of these proteins. Using this peptide, we demonstrate that disruption of the β-arrestin
1/ARHGAP21 complex results in a more active ARHGAP21, leading to less-efficient signaling via the angiotensin II type 1A receptor
and, thereby, attenuation of stimulated stress fiber formation.
Full-text · Article · Mar 2011 · Molecular and Cellular Biology
[Show abstract][Hide abstract] ABSTRACT: Enzymes from the PDE (phosphodiesterase) 4 cAMP-specific PDE family are crucial for the maintenance of compartmentalized cAMP responses in many cell types. Regulation of PDE activity can be achieved via post-translational modification such as phosphorylation by ERK (extracellular-signal-regulated kinase) MAPKs (mitogen-activated protein kinases) and PKA (protein kinase A). In the present paper, we report for the first time that PDE4 isoforms from the PDE4A and PDE4D subfamilies can be selectively modified by SUMO (small ubiquitin-related modifier). We have identified a single SUMO site within a consensus tetrapeptide motif, PsiKXE (where Psi represents a hydrophobic residue), which lies in the catalytic unit of these enzymes. SUMO modification of PDE4 at this site was observed upon overexpression of the SUMO E3 ligase PIASy [protein inhibitor of activated STAT (signal transducer and activator of transcription) Y] in HEK (human embryonic kidney)-293 cells and we identify PIASy as a novel binding partner for long PDE4 isoforms. Site-directed mutagenesis of the acceptor lysine residue ablated conjugation of PDE4 with SUMO, suggesting the presence of a single SUMO site in the first subdomain of the conserved PDE4 catalytic unit. This observation was supported by both cell-free in vitro SUMOylation assays and analysis of SUMOylated spot-immobilized peptide arrays. SUMO modification of long PDE4 isoforms serves to augment their activation by PKA phosphorylation and repress their inhibition by ERK phosphorylation. Following ligation of beta-adrenergic receptors, SUMOylation of PDE4 isoforms sufficiently amplified PKA-stimulated PDE4 activity to reduce markedly the PKA phosphorylation status of the beta2-adrenergic receptor. These results highlight a new means whereby cells might achieve the selective regulation of the activity of cAMP-specific PDE4 enyzmes.
Full-text · Article · Mar 2010 · Biochemical Journal
[Show abstract][Hide abstract] ABSTRACT: Beta arrestins are molecular scaffolds that can bring together three-component mitogen-activated protein kinase signalling modules to promote signal compartmentalisation. We use peptide array technology to define novel interfaces between components within the c-Jun N-terminal kinase (JNK)/beta arrestin signalling complex. We show that beta arrestin 1 and beta arrestin 2 associate with JNK3 via the kinase N-terminal domain in a region that, surprisingly, does not harbour a known 'common docking' motif. In the N-domain and C-terminus of beta arrestin 1 and beta arrestin 2 we identify two novel apoptosis signal-regulating kinase 1 binding sites and in the N-domain of the beta arrestin 1 and beta arrestin 2 we identify a novel MKK4 docking site.