Serine 58 of 14-3-3ζ Is a molecular switch regulating ASK1 and oxidant stress-induced cell death

Oxidant stress is a ubiquitous stressor with negative impacts on multiple cell types. ASK1 is a central mediator of oxidant injury, but while mechanisms of its inhibition, such as sequestration by 14-3-3 proteins and thioredoxin, have been identified, mechanisms of activation have remained obscure and the signaling pathways regulating this are not clear. Here, we report that phosphorylation of 14-3-3ζ at serine 58 (S58) is dynamically regulated in the cell and that the phosphorylation status of S58 is a critical factor regulating oxidant stress-induced cell death. Phosphorylation of S58 releases ASK1 from 14-3-3ζ, and ASK1 then activates stress-activated protein kinases, leading to cell death. While several members of the mammalian sterile 20 (Mst) family of kinases can phosphorylate S58 when overexpressed, we identify Ste20/oxidant stress response kinase 1 (SOK-1), an Mst family member known to be activated by oxidant stress, as a central endogenous regulator of S58 phosphorylation and thereby of ASK1-mediated cell death. Our findings identify a novel pathway that regulates ASK1 activation and oxidant stress-induced cell death

Interaction of 14-3-3 with Signaling Proteins Is Mediated by the Recognition of Phosphoserine

AbstractThe highly conserved and ubiquitously expressed 14-3-3 family of proteins bind to a variety of proteins involved in signal transduction and cell cycle regulation. The nature and specificity of 14-3-3 binding is, however, not known. Here we show that 14-3-3 is a specific phosphoserine-binding protein. Using a panel of phosphorylated peptides based on Raf-1, we have defined the 14-3-3 binding motif and show that most of the known 14-3-3 binding proteins contain the motif. Peptides containing the motif could disrupt 14-3-3 complexes and inhibit maturation of Xenopus laevis oocytes. These results suggest that the interactions of 14-3-3 with signaling proteins are critical for the activation of signaling proteins. Our findings also suggest novel roles for serine/threonine phosphorylation in the assembly of protein–protein complexes

RGS4 inhibits angiotensin II signaling and macrophage localization during renal reperfusion injury independent of vasospasm

Vascular inflammation is a major contributor to the severity of acute kidney injury. In the context of vasospasm-independent reperfusion injury we studied the potential anti-inflammatory role of the Gα-related RGS protein, RGS4. Transgenic RGS4 mice were resistant to 25 minute injury, although post-ischemic renal arteriolar diameter was equal to the wild type early after injury. A 10 minute unilateral injury was performed to study reperfusion without vasospasm. Eighteen hours after injury blood flow was decreased in the inner cortex of wild type mice with preservation of tubular architecture. Angiotensin II levels in the kidneys of wild type and transgenic mice were elevated in a sub-vasoconstrictive range 12 and 18 hours after injury. Angiotensin II stimulated pre-glomerular vascular smooth muscle cells (VSMC) to secrete the macrophage chemoattractant, RANTES; a process decreased by angiotensin II R2 (AT2) inhibition. However, RANTES increased when RGS4 expression was suppressed implicating Gα protein activation in an AT2-RGS4-dependent pathway. RGS4 function, specific to VSMC, was tested in a conditional VSMC-specific RGS4 knockout showing high macrophage density by T2 MRI compared to transgenic and non-transgenic mice after the 10 minute injury. Arteriolar diameter of this knockout was unchanged at successive time points after injury. Thus, RGS4 expression, specific to renal VSMC, inhibits angiotensin II-mediated cytokine signaling and macrophage recruitment during reperfusion, distinct from vasomotor regulation

14-3-3 proteins block apoptosis and differentially regulate MAPK cascades

14-3-3 family members are dimeric phosphoserine-binding proteins that participate in signal transduction and checkpoint control pathways. In this work, dominant-negative mutant forms of 14-3-3 were used to disrupt 14-3-3 function in cultured cells and in transgenic animals. Transfection of cultured fibroblasts with the R56A and R60A double mutant form of 14-3-3ζ (DN–14-3-3ζ) inhibited serum-stimulated ERK MAPK activation, but increased the basal activation of JNK1 and p38 MAPK. Fibroblasts transfected with DN–14-3-3ζ exhibited markedly increased apoptosis in response to UVC irradiation that was blocked by pre-treatment with a p38 MAPK inhibitor, SB202190. Targeted expression of DN–14-3-3η to murine postnatal cardiac tissue increased the basal activation of JNK1 and p38 MAPK, and affected the ability of mice to compensate for pressure overload, which resulted in increased mortality, dilated cardiomyopathy and massive cardiomyocyte apoptosis. These results demonstrate that a primary function of mammalian 14-3-3 proteins is to inhibit apoptosis

RGS4, a GTPase activator, improves renal function in ischemia–reperfusion injury

Acute kidney dysfunction after ischemia–reperfusion injury (IRI) may be a consequence of persistent intrarenal vasoconstriction. Regulators of G-protein signaling (RGSs) are GTPase activators of heterotrimeric G proteins that can regulate vascular tone. RGS4 is expressed in vascular smooth muscle cells in the kidney; however, its protein levels are low in many tissues due to N-end rule-mediated polyubiquitination and proteasomal degradation. Here, we define the role of RGS4 using a mouse model of IRI comparing wild-type (WT) with RGS4-knockout mice. These knockout mice were highly sensitized to the development of renal dysfunction following injury exhibiting reduced renal blood flow as measured by laser-Doppler flowmetry. The kidneys from knockout mice had increased renal vasoconstriction in response to endothelin-1 infusion ex vivo. The intrinsic renal activity of RGS4 was measured following syngeneic kidney transplantation, a model of cold renal IRI. The kidneys transplanted between knockout and WT mice had significantly reduced reperfusion blood flow and increased renal cell death. WT mice administered MG-132 (a proteasomal inhibitor of the N-end rule pathway) resulted in increased renal RGS4 protein and in an inhibition of renal dysfunction after IRI in WT but not in knockout mice. Thus, RGS4 antagonizes the development of renal dysfunction in response to IRI