Dataset Information

Src homology 2 domain containing protein 5 (SH2D5) binds the breakpoint cluster region protein, BCR, and regulates levels of Rac1-GTP.

ABSTRACT: SH2D5 is a mammalian-specific, uncharacterized adaptor-like protein that contains an N-terminal phosphotyrosine-binding domain and a C-terminal Src homology 2 (SH2) domain. We show that SH2D5 is highly enriched in adult mouse brain, particularly in Purkinjie cells in the cerebellum and the cornu ammonis of the hippocampus. Despite harboring two potential phosphotyrosine (Tyr(P)) recognition domains, SH2D5 binds minimally to Tyr(P) ligands, consistent with the absence of a conserved Tyr(P)-binding arginine residue in the SH2 domain. Immunoprecipitation coupled to mass spectrometry (IP-MS) from cultured cells revealed a prominent association of SH2D5 with breakpoint cluster region protein, a RacGAP that is also highly expressed in brain. This interaction occurred between the phosphotyrosine-binding domain of SH2D5 and an NxxF motif located within the N-terminal region of the breakpoint cluster region. siRNA-mediated depletion of SH2D5 in a neuroblastoma cell line, B35, induced a cell rounding phenotype correlated with low levels of activated Rac1-GTP, suggesting that SH2D5 affects Rac1-GTP levels. Taken together, our data provide the first characterization of the SH2D5 signaling protein.

SUBMITTER: Gray EJ

PROVIDER: S-EPMC4271225 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Src homology 2 domain containing protein 5 (SH2D5) binds the breakpoint cluster region protein, BCR, and regulates levels of Rac1-GTP.

Gray Elizabeth J EJ Petsalaki Evangelia E James D Andrew DA Bagshaw Richard D RD Stacey Melissa M MM Rocks Oliver O Gingras Anne-Claude AC Pawson Tony T

The Journal of biological chemistry 20141020 51

SH2D5 is a mammalian-specific, uncharacterized adaptor-like protein that contains an N-terminal phosphotyrosine-binding domain and a C-terminal Src homology 2 (SH2) domain. We show that SH2D5 is highly enriched in adult mouse brain, particularly in Purkinjie cells in the cerebellum and the cornu ammonis of the hippocampus. Despite harboring two potential phosphotyrosine (Tyr(P)) recognition domains, SH2D5 binds minimally to Tyr(P) ligands, consistent with the absence of a conserved Tyr(P)-bindin ...[more]

PMID: 25331951

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Project description:UnlabelledMicroRNAs (miRNAs) are small, ∼ 22-nucleotide-long RNAs that regulate gene expression posttranscriptionally. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes 12 pre-miRNAs during latency, and the functional significance of these microRNAs during KSHV infection and their cellular targets have been emerging recently. Using a previously reported microarray profiling analysis, we identified breakpoint cluster region mRNA (Bcr) as a cellular target of the KSHV miRNA miR-K12-6-5p (miR-K6-5). Bcr protein levels were repressed in human umbilical vein endothelial cells (HUVECs) upon transfection with miR-K6-5 and during KSHV infection. Luciferase assays wherein the Bcr 3' untranslated region (UTR) was cloned downstream of a luciferase reporter showed repression in the presence of miR-K6-5, and mutation of one of the two predicted miR-K6-5 binding sites relieved this repression. Furthermore, inhibition or deletion of miR-K6-5 in KSHV-infected cells showed increased Bcr protein levels. Together, these results show that Bcr is a direct target of the KSHV miRNA miR-K6-5. To understand the functional significance of Bcr knockdown in the context of KSHV-associated disease, we hypothesized that the knockdown of Bcr, a negative regulator of Rac1, might enhance Rac1-mediated angiogenesis. We found that HUVECs transfected with miR-K6-5 had increased Rac1-GTP levels and tube formation compared to HUVECs transfected with control miRNAs. Knockdown of Bcr in latently KSHV-infected BCBL-1 cells increased the levels of viral RTA, suggesting that Bcr repression by KSHV might aid lytic reactivation. Together, our results reveal a new function for both KSHV miRNAs and Bcr in KSHV infection and suggest that KSHV miRNAs, in part, promote angiogenesis and lytic reactivation.ImportanceKaposi's sarcoma (KS)-associated herpesvirus (KSHV) infection is linked to multiple human cancers and lymphomas. KSHV encodes small nucleic acids (microRNAs) that can repress the expression of specific human genes, the biological functions of which are still emerging. This report uses a variety of approaches to show that a KSHV microRNA represses the expression of the human gene called breakpoint cluster region (Bcr). Repression of Bcr correlated with the activation of a protein previously shown to cause KS-like lesions in mice (Rac1), an increase in KS-associated phenotypes (tube formation in endothelial cells and vascular endothelial growth factor [VEGF] synthesis), and modification of the life cycle of the virus (lytic replication). Our results suggest that KSHV microRNAs suppress host proteins and contribute to KS-associated pathogenesis.

Project description:BackgroundThe Src homology-2 domain protein B (Shb) is an adapter protein operating downstream of several tyrosine kinase receptors and consequently Shb regulates various cellular responses. Absence of Shb was recently shown to reduce hematopoietic stem cell proliferation through activation of focal adhesion kinase (FAK) and thus we sought to investigate Shb's role in the progression of leukemia.MethodsWild type and Shb knockout bone marrow cells were transformed with a retroviral BCR-ABL construct and subsequently transplanted to wild type or Shb knockout recipients. Disease latency, bone marrow and peripheral blood cell characteristics, cytokine expression, signaling characteristics and colony formation were determined by flow cytometry, qPCR, western blotting and methylcellulose colony forming assays.ResultsIt was observed that Shb knockout BCR-ABL-transformed bone marrow cells produced a disease with death occurring at earlier time points compared with corresponding wild type controls due to elevated proliferation of transformed bone marrow cells. Moreover, significantly elevated interleukin-6 and granulocyte colony-stimulation factor mRNA levels were observed in Shb knockout c-Kit + leukemic bone marrow cells providing a plausible explanation for the concurrent peripheral blood neutrophilia. Shb knockout leukemic bone marrow cells also showed increased ability to form colonies in methylcellulose devoid of cytokines that was dependent on the concomitantly observed increased activity of FAK. Transplanting BCR-ABL-transformed Shb knockout bone marrow cells to Shb knockout recipients revealed decreased disease latency without neutrophilia, thus implicating the importance of niche-derived cues for the increase of blood granulocytes.ConclusionsAbsence of Shb accelerates disease progression by exerting dual roles in BCR-ABL-induced leukemia: increased cell expansion due to elevated FAK activity and neutrophilia in peripheral blood, the latter dependent on the genetic background of the leukemic niche.

Dataset Information

Src homology 2 domain containing protein 5 (SH2D5) binds the breakpoint cluster region protein, BCR, and regulates levels of Rac1-GTP.

Publications

Src homology 2 domain containing protein 5 (SH2D5) binds the breakpoint cluster region protein, BCR, and regulates levels of Rac1-GTP.

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