Journal of Molecular Signaling最新文献

We have previously reported that stable expression of a dominant negative Death Receptor 5 (dnDR5) in skeletal myoblasts results in decreased basal caspase activity and decreased mRNA and protein expression of the muscle regulatory transcription factor MyoD in growth medium (GM), resulting in inhibited differentation when myoblasts are then cultured in differentiation media (DM). Further, this decreased level of MyoD mRNA was not a consequence of altered message stability, but rather correlated with decreased acetylation of histones in the distal regulatory region (DRR) of the MyoD extended promoter known to control MyoD transcription. As serum response factor (SRF) is the transcription factor known to be responsible for basal MyoD expression in GM, we compared the level of SRF binding to the non-canonical serum response element (SRE) within the DRR in parental and dnDR5 expressing myoblasts. Herein, we report that stable expression of dnDR5 resulted in decreased levels of serum response factor (SRF) binding to the CArG box in the SRE of the DRR. Total SRF expression levels were not affected, but phosphorylation indicative of SRF activation was impaired. This decreased SRF phosphorylation correlated with decreased phosphorylation-induced activation of p38 kinase. Moreover, the aforementioned signaling events affected by expression of dnDR5 could be appropriately recapitulated using either a pharmacological inhibitor of caspase 3 or p38 kinase. Thus, our results have established a signaling pathway from DR5 through caspases to p38 kinase activation, to SRF activation and the basal expression of MyoD.

Cellular signaling pathways are often interconnected. They accurately and efficiently regulate essential cell functions such as protein synthesis, cell growth, and survival. The target of rapamycin (TOR) signaling pathway and the endoplasmic reticulum (ER) stress response pathway regulate similar cellular processes. However, the crosstalk between them has not been appreciated until recently and the detailed mechanisms remain unclear. Here, we show that ER stress-inducing drugs activate the TOR signaling pathway in S2R+ Drosophila cells. Activating transcription factor 6 (Atf6), a major stress-responsive ER transmembrane protein, is responsible for ER stress-induced TOR activation. Supporting the finding, we further show that knocking down of both site-1/2 proteases (S1P/S2P), Atf6 processing enzymes, are necessary to connect the two pathways.

Myocyte Stress Protein 1 (MS1) is a muscle-specific, stress-responsive, regulator of gene expression. It was originally identified in embryonic mouse heart which showed increased expression in a rat model of left ventricular hypertrophy. To determine if MS1 was responsive to other stresses relevant to cardiac myocyte function, we tested if it could be induced by the metabolic stresses associated with ischaemia/reperfusion injury in cardiac myocytes. We found that metabolic stress increased MS1 expression, both at the mRNA and protein level, concurrent with activation of the c-Jun N-terminal Kinase (JNK) signalling pathway. MS1 induction by metabolic stress was blocked by both the transcription inhibitor actinomycin D and a JNK inhibitor, suggesting that activation of the JNK pathway during metabolic stress in cardiac myocytes leads to transcriptional induction of MS1. MS1 was also found to be an efficient JNK substrate in vitro, with a major JNK phosphorylation site identified at Thr-62. In addition, MS1 was found to co-precipitate with JNK, and inspection of the amino acid sequence upstream of the phosphorylation site, at Thr-62, revealed a putative Mitogen-Activated Protein Kinase (MAPK) binding site. Taken together, these data identify MS1 as a likely transcriptional and post-translational target for the JNK pathway in cardiac myocytes subjected to metabolic stress.

The Shc family of adaptor proteins is a group of proteins that lacks intrinsic enzymatic activity. Instead, Shc proteins possess various domains that allow them to recruit different signalling molecules. Shc proteins help to transduce an extracellular signal into an intracellular signal, which is then translated into a biological response. The Shc family of adaptor proteins share the same structural topography, CH2-PTB-CH1-SH2, which is more than an isoform of Shc family proteins; this structure, which includes multiple domains, allows for the posttranslational modification of Shc proteins and increases the functional diversity of Shc proteins. The deregulation of Shc proteins has been linked to different disease conditions, including cancer and Alzheimer's, which indicates their key roles in cellular functions. Accordingly, a question might arise as to whether Shc proteins could be targeted therapeutically to correct their disturbance. To answer this question, thorough knowledge must be acquired; herein, we aim to shed light on the Shc family of adaptor proteins to understand their intracellular role in normal and disease states, which later might be applied to connote mechanisms to reverse the disease state.

The ADP-ribosyltransferase C3 exoenzyme from C. botulinum selectively inactivates Rho and is therefore often used as an inhibitor for investigations on Rho signaling. Previous studies of our group revealed that C3 inhibited cell proliferation in HT22 cells accompanied by increased transcriptional activities of Sp1 and c-Jun and reduced levels of cyclin D1, p21 and phosphorylated p38. By use of a p38α-deficient and a p38α-expressing control cell line, the impact of p38 on C3-mediated inhibition of cell proliferation and alterations on MAPK signaling was studied by growth kinetic experiments and Western blot analyses. The cell growth of p38α-expressing cells was impaired by C3, while the p38α-deficient cells did not exhibit any C3-induced effect. The activity of the MKK3/6-p38 MAPK signaling cascade as well as the phosphorylation of c-Jun and JNK was reduced by C3 exclusively in the presence of p38α. Moreover, the activity of upstream MAPKKK TAK1 was lowered in the p38α-expressing cells. These results indicated a resistance of p38α-deficient cells to C3-mediated inhibition of cell growth. This anti-proliferative effect was highly associated with the decreased activity of c-Jun and upstream p38 and JNK MAPK signaling as a consequence of the absence of p38α in these cells.

Corticotropin-releasing factor (CRF) receptor1 (CRFR1) is associated with psychiatric illness and is a proposed target for the treatment of anxiety and depression. Similar to many G protein-coupled receptors (GPCRs), CRFR1 harbors a PDZ (PSD-95/Disc Large/Zona Occludens)-binding motif at the end of its carboxyl-terminal tail. The interactions of PDZ proteins with GPCRs are crucial for the regulation of receptor function. In the present study, we characterize the interaction of all members of the membrane-associated guanylate kinase with inverted orientation PDZ (MAGI) proteins with CRFR1. We show using co-immunoprecipitation that CRFR1 interacts with MAGI-1 and MAGI-3 in human embryonic kidney (HEK293) cells in a PDZ motif-dependent manner. We find that overexpression as well as knockdown of MAGI proteins result in a significant reduction in CRFR1 endocytosis. This effect is dependent on an intact PDZ binding motif for MAGI-2 and MAGI-3 but not MAGI-1. We show that the alteration in expression levels of MAGI-1, MAGI-2 or MAGI-3 can interfere with β-arrestin recruitment to CRFR1. This could explain the effects observed with receptor internalization. We also find that knockdown of endogenous MAGI-1, MAGI-2 or MAGI-3 in HEK293 cells can lead to an enhancement in ERK1/2 signaling but has no effect on cAMP formation. Interestingly, we observe a compensation effect between MAGI-1 and MAGI-3. Taken together, our data suggest that the MAGI proteins, MAGI-1, MAGI-2 and MAGI-3 can regulate β-arrestin-mediated internalization of CRFR1 as well as its signaling and that there is a compensatory mechanism involved in regulating the function of the MAGI subfamily.

Inositol hexakisphosphate kinase 2 (IP6K2) potentiates pro-apoptotic signalling and increases the sensitivity of mammalian cells to cytotoxic agents. Diphosphoinositol pentakisphosphate kinase (PPIP5K) generates inositol pyrophosphates (InsPPs) that are structurally distinct from those produced by IP6K2 and their possible roles in affecting cell viability remain unclear. In the present study, we tested the impact of PPIP5K1 on cellular sensitivity to various genotoxic agents to determine if PPIP5K1 and IP6K2 contribute similarly to apoptosis. We observed that PPIP5K1 overexpression decreased sensitivity of cells toward several cytotoxic agents, including etoposide, cisplatin, and sulindac. We further tested the impact of PPIP5K1 overexpression on an array of apoptosis markers and observed that PPIP5K1 decreased p53 phosphorylation on key residues, including Ser-15, -46, and -392. Overexpression of a kinase-impaired PPIP5K1 mutant failed to protect cells from apoptosis, indicating this protection is a consequence PPIP5K1 catalytic activity, in contrast with the sensitivity conferred by IP6K2, which is dependent on both catalytic and non-catalytic functions. These observations reveal distinct roles for PPIP5K1 and IP6K2 and the InsPPs they produce in controlling cell death.

AKAP-Lbc is a Rho-activating guanine nucleotide exchange factor (RhoGEF) important in heart development and pro-fibrotic signaling in cardiomyocytes. Heterotrimeric G proteins of the G12/13 subfamily, comprising Gα12 and Gα13, are well characterized as stimulating a specialized group of RhoGEFs through interaction with their RGS-homology (RH) domain. Despite lacking an RH domain, AKAP-Lbc is bound by Gα12 through an unknown mechanism to activate Rho signaling. We identified a Gα12-binding region near the C-terminus of AKAP-Lbc, closely homologous to a region of p114RhoGEF that we also discovered to interact with Gα12. This binding mechanism is distinct from the well-studied interface between RH-RhoGEFs and G12/13 α subunits, as demonstrated by Gα12 mutants selectively impaired in binding either this AKAP-Lbc/p114RhoGEF region or RH-RhoGEFs. AKAP-Lbc and p114RhoGEF showed high specificity for binding Gα12 in comparison to Gα13, and experiments using chimeric G12/13 α subunits mapped determinants of this selectivity to the N-terminal region of Gα12. In cultured cells expressing constitutively GDP-bound Gα12 or Gα13, the Gα12 construct was more potent in exerting a dominant-negative effect on serum-mediated signaling to p114RhoGEF, demonstrating coupling of these signaling proteins in a cellular pathway. In addition, charge-reversal of conserved residues in AKAP-Lbc and p114RhoGEF disrupted Gα12 binding for both proteins, suggesting they harbor a common structural mechanism for interaction with this α subunit. Our results provide the first evidence of p114RhoGEF as a Gα12 signaling effector, and define a novel region conserved between AKAP-Lbc and p114RhoGEF that allows Gα12 signaling input to these non-RH RhoGEFs.