Role of PH domain of BCR/ABL fusion protein in cytoskeleton remodeling.
In: Biopolymers & Cell, 2012-12-02, S. 6-6
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Background: Reciprocal translocation between short arms of 9 and 22 chromosomes leads to generation of fusion gene BCR-ABL. To date, three forms of such fusion genes were identified and named according to molecular weight of their products: p185, p210, and p230. Difference in molecular weights is due to the absence or presence of certain domains of BCR protein, such as Pleckstrin-homology (PH) and Dbl-homology (DH) domains. The different forms of BCR-ABL protein are associated with different myeloproliferative disorders: p185 - with acute lymphoblastic leukemia, p210 - with chronic myelogenous leukemia, and p230 - with true thrombocytopenia. A shorter form of BCR-ABL leads to a more aggressive phenotype of disorder, while p210 and p230 lead to more mild and slow development. Therefore, it is important to determine the role of above mentioned domains in signaling and regulatory cellular pathways. According to the earlier research of Miroshnichenko D. et. al. 23 proteinprotein interactions were identified between DH-PH part of BCR and various cellular proteins of K562 cell line. Among them, several may be grouped by functional similarity. For instance, cortactin (CTTN), cytokeratin type 10 (KRT10), tubulin beta 1 chain (TUBB1), and Collagen IV alpha-1 polypeptide (COL4A1) are involved in cytoskeletal remodeling, membrane signaling, and cell adhesion. As BCR-ABL p210 is associated with abnormal cytoskeletal behavior, altered adhesion, and localization on cell cortex, a role of PH domain in these processes should be determined and previous data on protein-protein interactions verified by more sensitive methods. Aim. To determine cellular localization and protein-protein interactions of PH domain of BCR protein with COL4A1, CTTN, KRT10, and TUBB1 proteins. Methods. pCMV-SPORT6- based constructs with full coding sequences of COL4A1, KRT10, and TUBB1 were acquired from Open Biosystems. Construct with full CTTN sequence was kindly provided by Pontus Aspenstrom (Karolinska Institute, Sweden). CTTN and TUBB1 were amplified by PCR with specific primers and ligated to pBluescriptSKII(+) on EcoRI-BamHI and EcoRV sites, respectively. Further, CTTN was subligated to pGEX4T2 on EcoRI-NotI, to pCMV-myc on EcoRIBglII, and pECFP-C3 on EcoRI-BamHI. TUBB1 was subligated to pGEX4T3 on SalI-NotI, pcDNA4HisMaxC on EcoRV-NotI, and pECFP-C3 on HindIIIEcoRI. Coding sequence of COL4A1 was cut from pCMVSPORT6 on AscIHindIII and ligated to pECFP-C1 on XhoI(blunted)-HindIII sites. KRT10 was subligated from pCMVSPORT6 on EcoRI-NotI to pGEX4T2 and pCMV-HA, and on EcoRI-SalI to pECFP-C3. Results. The genetic constructs for bacterial and mammalian expression were created and verified. Conclusions. The derived vectors are suitable for further protein production and consequent protein-protein interaction analysis by far-Western blot, Coimmunoprecipitation and FRET. [ABSTRACT FROM AUTHOR]
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Role of PH domain of BCR/ABL fusion protein in cytoskeleton remodeling.
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Autor/in / Beteiligte Person: | Gurianov, D. S. ; Kravchuk, I. V. ; Maliuta, O. V. ; Dybkov, M. V. ; Telegeev, G. D. |
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Zeitschrift: | Biopolymers & Cell, 2012-12-02, S. 6-6 |
Veröffentlichung: | 2012 |
Medientyp: | academicJournal |
ISSN: | 0233-7657 (print) |
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