p160ROCK mediates RhoA activation of Na-H exchange.

The ubiquitously expressed Na-H exchanger, NHE1, acts downstream of RhoA in a pathway regulating focal adhesion and actin stress fiber formation. p160ROCK, a serine/threonine protein kinase, is a direct RhoA target mediating RhoA-induced assembly of focal adhesions and stress fibers. Here, stress fiber formation induced by p160ROCK was inhibited by the addition of a specific NHE1 inhibitor, ethylisopropylamiloride, in CCL39 fibroblasts, and was absent in PS120 mutant fibroblasts lacking NHE1. In CCL39 cells, NHE1 activity was stimulated by expression of mutationally active p160ROCK, but not by mutationally active protein kinase N, another RhoA target kinase. Expression of a dominant interfering p160ROCK inhibited RhoA-, but not Cdc42- or Rac-activation of NEH1. In addition, the p160ROCK-specific inhibitor Y-27632 inhibited increases in NHE1 activity in response to RhoA, and to lysophosphatidic acid (LPA), which stimulates RhoA, and it also inhibited LPA-increased phosphorylation of NHE1. A C-terminal truncation of NHE1 abolished both LPA-induced phosphorylation and activation of the exchanger. Furthermore, mutationally active p160ROCK phosphorylated an NHE1 C-terminal fusion protein in vitro, and this was inhibited in the presence of Y-27632. Phosphopeptide maps indicated that identical residues in NHE1 were phosphorylated by p160ROCK in vivo and in vitro. These findings identify p160ROCK as an upstream, possibly direct, activator of NHE1, and suggest that NHE1 activity and phosphorylation are necessary for actin stress fiber assembly induced by p160ROCK.     

RhoA/ROCK downregulates FPR2-mediated NADPH oxidase activation in mouse bone marrow granulocytes

Polymorphonuclear neutrophils (PMNs) express the high and low affinity receptors to formylated peptides (mFPR1 and mFPR2 in mice, accordingly). RhoA/ROCK (Rho activated kinase) pathway is crucial for cell motility and oxidase activity regulated via FPRs. There are contradictory data on RhoA-mediated regulation of NADPH oxidase activity in phagocytes. We have shown divergent Rho GTPases signaling via mFPR1 and mFPR2 to NADPH oxidase in PMNs from inflammatory site. The present study was aimed to find out the role of RhoA/ROCK in the respiratory burst activated via mFPR1 and mFPR2 in the bone marrow PMNs. Different kinetics of RhoA activation were detected with 0.1 μM fMLF and 1 μM WKYMVM operating via mFPR1 and mFPR2, accordingly. RhoA was translocated in fMLF-activated cells towards the cell center and juxtamembrane space versus uniform allocation in the resting cells. Specific inhibition of RhoA by CT04, Rho inhibitor I, weakly depressed the respiratory burst induced via mFPR1, but significantly increased the one induced via mFPR2. Inhibition of ROCK, the main effector of RhoA, by Y27632 led to the same effect on the respiratory burst. Regulation of mFPR2-induced respiratory response by ROCK was impossible under the cytoskeleton disruption by cytochalasin D, whereas it persisted in the case of mFPR1 activation. Thus we suggest RhoA to be one of the regulatory and signal transduction components in the respiratory burst through FPRs in the mouse bone marrow PMNs. Both mFPR1 and mFPR2 binding with a ligand trigger the activation of RhoA. FPR1 signaling through RhoA/ROCK increases NADPH-oxidase activity. But in FPR2 action RhoA/ROCK together with cytoskeleton-linked systems down-regulates NADPH-oxidase. This mechanism could restrain the reactive oxygen species dependent damage of own tissues during the chemotaxis of PMNs and in the resting cells.     

Human papillomavirus E6 proteins bind p53 in vivo and abrogate p53-mediated repression of transcription.

The transforming proteins of DNA tumor viruses SV40, adenovirus and human papillomaviruses (HPV) bind the retinoblastoma and p53 cell cycle regulatory proteins. While the binding of SV40 large T antigen and the adenovirus E1B 55 kDa protein results in the stabilization of the p53 protein, the binding of HPV16 and 18 E6 results in enhanced degradation in vitro. To explore the effect of viral proteins on p53 stability in vivo, we have examined cell lines immortalized in tissue culture by HPV18 E6 and E7 or SV40 large T antigen, as well as cell lines derived from cervical neoplasias. The half-life of the p53 protein in non-transformed human foreskin keratinocytes in culture was found to be approximately 3 h while in cell lines immortalized by E6 and E7, p53 protein half-lives ranged from 2.8 h to less than 1 h. Since equivalent levels of E6 were found in these cells, the range in p53 levels observed was not a result of variability in amounts of E6. In keratinocyte lines immortalized by E7 alone, the p53 half-life was found to be similar to that in non-transformed cells; however, it decreased to approximately 1 h following supertransfection of an E6 gene. These observations are consistent with an interaction of E6 and p53 in vivo resulting in reductions in the stability of p53 ranging between 2- and 4-fold. We also observed that the expression of various TATA containing promoters was repressed in transient assays by co-transfection with plasmids expressing the wild-type p53 gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Beta-HPV 5 and 8 E6 promote p300 degradation by blocking AKT/p300 association

The E6 oncoprotein from high-risk genus alpha human papillomaviruses (α-HPVs), such as HPV 16, has been well characterized with respect to the host-cell proteins it interacts with and corresponding signaling pathways that are disrupted due to these interactions. Less is known regarding the interacting partners of E6 from the genus beta papillomaviruses (β-HPVs); however, it is generally thought that β-HPV E6 proteins do not interact with many of the proteins known to bind to α-HPV E6. Here we identify p300 as a protein that interacts directly with E6 from both α- and β-HPV types. Importantly, this association appears much stronger with β-HPV types 5 and 8-E6 than with α-HPV type 16-E6 or β-HPV type 38-E6. We demonstrate that the enhanced association between 5/8-E6 and p300 leads to p300 degradation in a proteasomal-dependent but E6AP-independent manner. Rather, 5/8-E6 inhibit the association of AKT with p300, an event necessary to ensure p300 stability within the cell. Finally, we demonstrate that the decreased p300 protein levels concomitantly affect downstream signaling events, such as the expression of differentiation markers K1, K10 and Involucrin. Together, these results demonstrate a unique way in which β-HPV E6 proteins are able to affect host-cell signaling in a manner distinct from that of the α-HPVs.