Molecular evolution of two duplicated CDPK genes CPK7 and CPK12 in grass species: a case study in wheat (Triticum aestivum L.)

Gene duplication contributes to the expansion of gene families and subsequent functional diversification. Calcium-dependent protein kinases (CDPKs) are members of an important calcium sensor family involved in abiotic and biotic stress signaling in plants. We report here the molecular evolution and expression analysis of a pair of duplicated CDPK genes CPK7 and CPK12 that arose in the common ancestor of grass species. With higher nonsynonymous/synonymous ratios (dN/dS, or ω), CPK12 genes appear to diverge more rapidly than CPK7s, suggesting relaxed selection constraints on CPK12s. Sliding window analysis revealed increased dN and ω values at N-terminal regions and the calcium-binding EF hand loops. Likelihood analyses using various models in PAML 4.0 showed purifying selection on both CPK7 and CPK12 lineages. In addition to the divergence in cis-element combinations on their promoters, functional divergence of CPK7 and CPK12 genes was also observed in wheat where TaCPK7 was found to respond to drought (PEG), salt (NaCl), cold, and hydrogen peroxide (H(2)O(2)) while TaCPK12 responded only to the treatment of ABA, a feature that may complement or expand TaCPK7-mediated stress signaling networks of wheat. The contrasting expression patterns of CPK7 and CPK12 genes under stress conditions were also observed in rice, suggesting conservative functional evolution of these genes. Since no positive selection was detected between the two lineages, the divergence of CPK7 and CPK12 genes should be ascribed to subfunctionalization, rather than neofunctionalization. Thus, our work demonstrates another case of evolutionary employment of duplicated genes via subfunctionalization for better adaptation.     

Point mutated caveolin-3 form (P104L) impairs myoblast differentiation via Akt and p38 signalling reduction, leading to an immature cell signature

Unbalanced levels of caveolin-3 (Cav3) are involved in muscular disorders. In the present study we show that differentiation of immortalized myoblasts is affected by either lack or overexpression of Cav3. Nevertheless, depletion of Cav3 induced by delivery of the dominant-negative Cav3 (P104L) form elicited a more severe phenotype, characterized by the simultaneous attenuation of the Akt and p38 signalling networks, leading to an immature cell and molecular signature. Accordingly, differentiation of myoblasts harbouring Cav3 (P104L) was improved by countering the reduced Akt and p38 signalling network via administration of IGF-1 or trichostatin A. Furthermore, loss of Cav3 correlated with a deregulation of the TGF-β-induced Smad2 and Erk1/2 pathways, confirming that Cav3 controls TGF-β signalling at the plasma membrane. Overall, these data suggest that loss of Cav3, primarily causing attenuation of both Akt and p38 pathways, contributes to impair myoblast fusion.     

ROS-activated p38 MAPK/ERK-Akt cascade plays a central role in palmitic acid-stimulated hepatocyte proliferation

In the past years, free fatty acids (FFAs) and obesity have been reported to play an important role in cancer development. Palmitic acid (PA) is the most prevalent saturated FFA in circulation. However, the mechanism underlying the effect of PA on cell proliferation is still to be elucidated. In this report, we, for the first time, investigate the signaling pathway in human normal hepatocytes (QZG) responsible for PA-induced proliferation. The results demonstrate that PA promotes cell cycle progression, accelerates cell proliferation, and induces a transient and sequential activation of a series of kinases. The employment of several inhibitors and antioxidants indicates that a ROS-induced stress-sensitive p38 MAPK/ERK-Akt cascade plays a critical role in the regulation of PA on cell cycle and cell proliferation. Moreover, PA dose and time dependently activates Nrf2 and this activation relies on ROS-induced stimulation of p38 MAPK/ERK-Akt signaling, demonstrating that Nrf2 activation may be associated with the regulation of PA on cell cycle transition and proliferation. In conclusion, our study elucidates the importance of PA metabolism on cell proliferation, and suggests that PA stimulates hepatocyte proliferation through activating the ROS-p38 MAPK/ERK-Akt cascade which is intersected with the activation of Nrf2 and that the effect of ROS on signal transduction is in a dose- and time-dependent manner. All the above noted provide a new clue for the central role of ROS in cell proliferation and tumorigenesis.     

ERK1 and ERK2 kinases activate hydroxyurea-induced S-phase checkpoint in MCF7 cells by mediating ATR activation

Modulation of MEK has been demonstrated to affect hydroxyurea (HU) induced-DNA damage response (DDR), implying the involvement of ERK1 and ERK2 in the process. To directly examine how the ERK kinases function in HU-initiated DDR, we knocked-down either ERK1 or ERK2 in MCF7 cells. This resulted in reduction of HU-induced phosphorylation of CHK1 S345 (serine 345), p53 S15, and H2AX S139. While HU potently induced CDC2 Y15 (tyrosine 15) phosphorylation, an event causing CDC2 inactivation, inhibition of ERK kinases using U0126 (a MEK inhibitor), MEK1K97M (a dominant negative MEK1), and knockdown of either ERK1 or ERK2 significantly attenuated HU-induced CDC2 Y15 phosphorylation. As CDC2 kinase activity is required for mitosis, our observations reveal that ERK1 and ERK2 kinases play important roles in preventing mitotic entry in response to HU. Consistent with ATR being the apical kinase to initiate HU-induced DDR, knockdown of ERK1 or ERK2 significantly inhibited HU-induced ATR recruitment to the stalled replication forks (ATR foci), an event required for ATR activation. Mechanistically, knockdown of ERK1 or ERK2 resulted in relocation of ATR from the nucleoplasm to the nucleolus in response to HU, therefore making ATR unavailable to the sites of DNA damage. Taken together, we demonstrate that ERK kinases sit upstream of ATR to facilitate its activation.     

iNOS/NO signaling regulates apoptosis induced by glycochenodeoxycholate in hepatocytes

Inducible nitric oxide synthase (iNOS) and nitric oxide (NO) can ameliorate apoptosis induced by toxic glycochenodeoxycholate (GCDC) in hepatocytes. However, the underlying molecular mechanisms are not yet understood in detail. This study is to clarify the function of iNOS/NO and its mechanisms during the apoptotic process. The apoptosis was brought about by GCDC in rat primary hepatocytes. iNOS/NO signaling was then investigated. iNOS inhibitor 1400 W enhanced the GCDC-induced apoptosis as reflected by caspase-3 activity and TUNEL assay. Exogenous NO regulated the apoptosis subsequent to NO donor S-nitroso-N-acetyl-penicillamine (SNAP) or sodium nitroprusside (SNP). The GCDC-induced apoptosis was decreased with 0.1 mM SNAP or 0.15 mM SNP, while it was increased with 0.8 mM SNAP or 1.2 mM SNP. The endogenous iNOS inhibited apoptosis, but the exogenous NO played a dual role during the GCDC-induced apoptosis. There was a potential iNOS/Akt/survivin axis that inhibited the hepatocyte apoptosis in low doses of NO donors. In contrast, high doses of NO donors activated CHOP through p38MAP-kinase (p38MAPK), upregulated TRAIL receptor DR5, and suppressed survivin. Consequently the high doses of NO donors promoted the apoptosis in hepatocytes. Our data suggest that the iNOS/NO signaling can modulate Akt/survivin and p38MAPK/CHOP pathways to mediate the GCDC-induced the apoptosis in hepatocytes. These signaling pathways may serve as targets for therapeutic intervention in cholestatic liver disease.     

Inhibition of c-Kit, VEGFR-2 (KDR), and ABCG2 by analogues of OSI-930

The quinoline domain of OSI-930, a dual inhibitor of receptor tyrosine kinases (RTKs) c-Kit and KDR, was modified in an effort to further understand the SAR of OSI-930, and the binding site characteristics of c-Kit and KDR. A series of 16 compounds with heteroatom substituted pyridyl and phenyl ring systems was synthesized and evaluated against a panel of kinases including c-Kit and KDR. Aminopyridyl derivative 6 was found to be the most active member of the series with 91% and 57% inhibition of c-Kit at 10μM and 1μM, respectively and 88% and 50% inhibition of KDR at 10μM and 1μM, respectively. The target compounds were also tested for their ability to inhibit efflux of mitoxantrone through inhibition of ATP dependent ABCG2 pump. Nitropyridyl derivative 5 and o-nitrophenyl derivative 7 exhibited complete inhibition of the ABCG2 pump with IC(50) values of 13.67μM and 16.67μM, respectively.     

Sensitive luciferin derived probes for selective carboxypeptidase activity

Highly selective luminescent probes, QLUC-TYR and LUC-GLU, for detection of carboxypeptidase activity were synthesized. Caged substrates were first cleaved by corresponding carboxypeptidases, and then they were activated by luciferase to emit light. Enzymatic activities of biologically important carboxypeptidases can be determined using this technology.     

Vascular endothelial growth factor receptor 2 direct interaction with nephrin links VEGF-A signals to actin in kidney podocytes

The transmembrane protein nephrin is an essential component of slit diaphragms, the specialized cell junctions that link podocyte foot processes. Podocytes are epithelial cells that surround the glomerular capillaries in the kidney and are necessary for the organ-filtering function. Nephrin signaling complex transduces extracellular cues to the podocyte cytoskeleton and regulates podocyte shape and function. Vascular endothelial growth factor A (VEGF-A) is a required growth factor produced and secreted by podocytes. Accumulating evidence suggests a cross-talk between VEGF-A and nephrin signaling pathways. We previously showed that in vivo nephrin associates with VEGF receptor-2 (VEGFR2), the signaling receptor for VEGF-A. In the present work, we characterized the interaction between nephrin and VEGFR2 in cultured cells and in vitro. We demonstrate that nephrin-VEGFR2 interaction is direct using mass spectrometry, immunoprecipitation, GST-binding assays, and blot overlay experiments. This interaction occurs through VEGFR2 and nephrin cytoplasmic domains. Nephrin-VEGFR2 interaction is modulated by tyrosine phosphorylation of both cytoplasmic domains. Furthermore, the nephrin-VEGFR2 complex involves Nck and actin. VEGF-A signaling via this complex results in decreased cell size. We provide evidence that this multiprotein interaction occurs in cultured podocytes. We propose that the nephrin-VEGFR2 complex acts as a key mediator to transduce local VEGF-A signals to the podocyte actin cytoskeleton, regulating the foot process structure and glomerular filter integrity.     

Fibroblast growth factor receptor 3 (FGFR3) is a strong heat shock protein 90 (Hsp90) client: implications for therapeutic manipulation

Fibroblast growth factor receptor 3 (FGFR3) is a key regulator of growth and differentiation, whose aberrant activation causes a number of genetic diseases including achondroplasia and cancer. Hsp90 is a specialized molecular chaperone involved in stabilizing a select set of proteins termed clients. Here, we delineate the relationship of Hsp90 and co-chaperone Cdc37 with FGFR3 and the FGFR family. FGFR3 strongly associates with these chaperone complexes and depends on them for stability and function. Inhibition of Hsp90 function using the geldanamycin analog 17-AAG induces the ubiquitination and degradation of FGFR3 and reduces the signaling capacity of FGFR3. Other FGFRs weakly interact with these chaperones and are differentially influenced by Hsp90 inhibition. The Hsp90-related ubiquitin ligase CHIP is able to interact and destabilize FGFR3. Our results establish FGFR3 as a strong Hsp90 client and suggest that modulating Hsp90 chaperone complexes may beneficially influence the stability and function of FGFR3 in disease.