Functional characterization of a novel RhoGAP protein Deleted in Liver Cancer 2 (DLC2)

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Selective STAT3-alpha or -beta expression reveals spliceform-specific phosphorylation kinetics, nuclear retention and distinct gene expression outcomes

Phosphorylation of STAT3 (signal transducer and activator of transcription 3) is critical for its nuclear import and transcriptional activity. Although a shorter STAT3β spliceform was initially described as a negative regulator of STAT3α, gene knockout studies have revealed that both forms play critical roles. We have expressed STAT3α and STAT3β at comparable levels to facilitate a direct comparison of their functional effects, and have shown their different cytokine-stimulated kinetics of phosphorylation and nuclear translocation. Notably, the sustained nuclear translocation and phosphorylation of STAT3β following cytokine exposure contrasted with a transient nuclear translocation and phosphorylation of STAT3α. Importantly, co-expression of the spliceforms revealed that STAT3β enhanced and prolonged the phosphorylation and nuclear retention of STAT3α, but a STAT3β R609L mutant, with a disrupted SH2 (Src homology 2) domain, was not tyrosine phosphorylated following cytokine stimulation and could not cross-regulate STAT3α. The physiological importance of prolonged phosphorylation and nuclear retention was indicated by transcriptome profiling of STAT3(-/-) cells expressing either STAT3α or STAT3β, revealing the complexity of genes that are up- and down-regulated by the STAT3 spliceforms, including a distinct set of STAT3β-specific genes regulated under basal conditions and after cytokine stimulation. These results highlight STAT3β as a significant transcriptional regulator in its own right, with additional actions to cross-regulate STAT3α phosphorylation and nuclear retention after cytokine stimulation

Dynamic microtubule association of Doublecortin X (DCX) is regulated by its C-terminus

Doublecortin X (DCX), known to be essential for neuronal migration and cortical layering in the developing brain, is a 40 kDa microtubule (MT)-associated protein. DCX directly interacts with MTs via its two structured doublecortin (DC) domains, but the dynamics of this association and the possible regulatory roles played by the flanking unstructured regions remain poorly defined. Here, we employ quantitative fluorescence recovery after photobleaching (FRAP) protocols in living cells to reveal that DCX shows remarkably rapid and complete exchange within the MT network but that the removal of the C-terminal region significantly slows this exchange. We further probed how MT organization or external stimuli could additionally modulate DCX exchange dynamics. MT depolymerisation (nocodazole treatment) or stabilization (taxol treatment) further enhanced DCX exchange rates, however the exchange rates for the C-terminal truncated DCX protein were resistant to the impact of taxol-induced stabilization. Furthermore, in response to a hyperosmotic stress stimulus, DCX exchange dynamics were slowed, and again the C-terminal truncated DCX protein was resistant to the stimulus. Thus, the DCX dynamically associates with MTs in living cells and its C-terminal region plays important roles in the MT-DCX association

Differences in c-Jun N-terminal kinase recognition and phosphorylation of closely related stathmin-family members

The fulltext of this publication will be made publicly available after relevant embargo periods have lapsed and associated copyright clearances obtained.The stathmin (STMN) family of tubulin-binding phosphoproteins are critical regulators of interphase microtubule dynamics and organization in a broad range of cellular processes. c-Jun N-terminal kinase (JNK) signalling to STMN family proteins has been implicated specifically in neuronal maturation, degeneration and cell stress responses more broadly. Previously, we characterized mechanisms underlying JNK phosphorylation of STMN at proline-flanked serine residues (Ser25 and Ser38) that are conserved across STMN-like proteins. In this study, we demonstrated using in vitro kinase assays and alanine replacement of serine residues that JNK phosphorylated the STMN-like domain (SLD) of SCG10 on Ser73, consistent with our previous finding that STMN Ser38 was the primary JNK target site. In addition, we confirmed that a JNK binding motif ((41)KKKDLSL(47)) that facilitates JNK targeting of STMN is conserved in SCG10. In contrast, SCLIP was phosphorylated by JNK primarily on Ser60 which corresponds to Ser25 on STMN. Moreover, although the JNK-binding motif identified in STMN and SCG10 was not conserved in SCLIP, JNK phosphorylation of SCLIP was inhibited by a substrate competitive peptide (TI-JIP) highlighting kinase-substrate interaction as required for JNK targeting. Thus, STMN and SCG10 are similarly targeted by JNK but there are clear differences in JNK recognition and phosphorylation of the closely related family member, SCLIP

Characterization of novel putative tumor suppressor gene, DLC2 (deleted in liver cancer 2), in hepatocellular carcinoma

Deleted in liver cancer 2 (DLC2) gene, a putative tumour suppressor gene located at chromosome 13q12.3, was first identified by our group (Ching et al., J Biol Chem 2003;278:10824-10830). The DLC2 gene has striking homology to another tumor suppressor gene, DLC1, located at chromosome 8p22-8p21.3. DLC2 encodes a Rho GTPase activating protein (RhoGAP) with GAP activity specific for RhoA and Cdc42. The chromosomal region of the DLC2 gene shows frequent deletion with loss of heterozygosity in human hepatocellular carcinoma (HCC). By RT-PCR analysis, DLC2 is also frequently underexpressed in HCCs compared with the corresponding nontumorous livers. These suggest that the DLC2 gene may play a role in liver carcinogenesis. In this study, we aimed to further characterize DLC2 biochemically and functionally. From the bioinformatics search of DLC2 gene in humans, we found that there were four different isoforms of DLC2 gene, which we designate as DLC2α, β, γ, and δ. The four isoforms of DLC2 gene were identified in silico. With RT-PCR using four pairs of isoform specific primers performed on 10 HCC cell lines, DLC2α, γ, and δ were found to be expressed in all hepatoma cell lines tested, with different levels of expression. In contrast, the mRNA expression of the DLC2β isoform could be detected in only 4 of these 10 HCC cell lines. Direct sequencing of the PCR products of these isoforms confirmed the presence of these isoforms. In addition, we analyzed the localization and the in vivo function of DLC2 gene. Transfection of DLC2α and DLC2γ into Swiss3T3 cells showed their predominant cytoplasmic localization. Interestingly, over-expression of DLC2α and DLC2γ induced morphological alterations in the transfected Swiss3T3 cells. The shape of transfected Swiss3T3 cells was changed from angular and spindle to round when compared with the non-transfected parent cells. Confocal microscopy revealed that the morphological changes were associated with remarkable inhibition of actin stress fiber formation. In conclusion, these results were in keeping with the functions of DLC2 as a RhoGAP and that it induced morphological alterations through regulation of cytoskeletal reorganization. DLC2 likely exerts its tumor suppressor function partly via its RhoGAP function to inhibit RhoA activity, resulting in suppression of stress fiber formation. Further studies on the functional characterization of the different isoforms of DLC2 may give insight into its tumor suppressor function

Identification of multiple isoforms of DLC2, a novel tumor suppressor gene, and their functional characterization in hepatocellular carcinoma

Young Investigator AwardConference Theme: Eastern and Western Experience

Mitochondrial targeting of growth suppressor protein DLC2 through the START domain

Deleted in liver cancer 2 (DLC2) is a candidate tumor suppressor frequently found to be deleted in hepatocellular carcinoma. In this study, we determined the subcellular localization of DLC2. Co-localization and biochemical fractionation studies revealed that DLC2 localized to mitochondria. In addition, the DLC2-containing cytoplasmic speckles were in proximity to lipid droplets. A DLC2 mutant containing the steroidogenic acute regulatory protein-related lipid transfer (START) domain only showed a localization pattern identical to that of DLC2. Taken together, we have provided the first evidence for mitochondrial localization of DLC2 through the START domain. These findings might have implications in liver physiology and carcinogenesis. © 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.link_to_subscribed_fulltex

Deleted in liver cancer (DLC) 2 encodes a RhoGAP protein with growth suppressor function and is underexpressed in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a major malignancy in many parts of the world, especially in Asia and Africa. Loss of heterozygosity (LOH) on the long arm of chromosome 13 has been reported in HCC. In search of tumor suppressor genes in this region, here we have identified DLC2 (for deleted in liver cancer 2) at 13q12.3 encoding a novel Rho family GTPase-activating protein (GAP). DLC2 mRNA is ubiquitously expressed in normal tissues but was significantly underexpressed in 18% (8/ 45) of human HCCs. DLC2 is homologous to DLC1, a previously identified tumor suppressor gene at 8p22p21.3 frequently deleted in HCC. DLC2 encodes a novel protein with a RhoGAP domain, a SAM (sterile α motif) domain related to p73/p63, and a lipid-binding STAR-related lipid transfer (START) domain. Biochemical analysis indicates that DLC2 protein has GAP activity specific for small GTPases RhoA and Cdc42. Expression of the GAP domain of DLC2 sufficiently inhibits the Rho-mediated formation of actin stress fibers. Introduction of human DLC2 into mouse fibroblasts suppresses Ras signaling and Ras-induced cellular transformation in a GAP-dependent manner. Taken together, our findings suggest a role for DLC2 in growth suppression and hepatocarcinogenesis.link_to_OA_fulltex