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HRPAP20: A NOVEL CALMODULIN-BINDING PHOSPHOPROTEIN INVOLVED IN TUMOR PROGRESSION

SHUKLA, MANASI NARENDRA
http://rave.ohiolink.edu/etdc/view?acc_num=ucin1175629479

Abstract Details

2007, PhD, University of Cincinnati, Pharmacy : Pharmaceutical Sciences.
We previously showed that HRPAP20, a novel PRL-regulated phosphoprotein, augmented proliferation and survival in rat Nb2 lymphoma and human MCF-7 breast cancer cell lines (Cancer Res. 64:1016, 2004). Our results indicate that malignant human cell lines MDA-MB-231 and MDA-MB-435, and invasive breast tumor specimens from patients express elevated levels of HRPAP20. Constitutive expression of HRPAP20 in weakly invasive MCF-7 cells enhanced their invasiveness, implicating that HRPAP20 expression may correlate with advanced disease. Using CaM-pulldown assays and co-IP analyses, we showed that HRPAP20 interacts with Ca2+ CaM in vitro and in quiescent Nb2 and MCF-7 cells. Moreover, observations from site-directed mutagenesis of the CaM-binding domain of HRPAP20 suggested an upstream requirement of the HRPAP20:CaM complex in MCF-7 cell invasion. (Oncogene. Mar 15, 1780). EGF and TPA caused a rapid disruption of the HRPAP20:CaM complex in MCF-7 cells. PRL treatment in Nb2 and PRL-deficient MCF-7 cells showed a similar pattern of HRPAP20:CaM dissociation. Together, these results suggested that the complex was regulated by these stimuli, all of which activate PKC. Furthermore, we showed that HRPAP20 is a substrate for PKC immunoprecipitated from EGF or PRL-stimulated MCF-7 cells. The effect of EGF on HRPAP20 phosphorylation was abolished by rottlerin, which inhibits PKC-δ; but not by Gö6976, an inhibitor of conventional PKC isoforms. Results from experiments employing dominant negative PKC-δ suggested that it may catalyze HRPAP20 phosphorylation, and may influence its effect on tumor cell invasion. On the other hand, both rottlerin and Gö6976 failed to inhibit PRL-mediated increase in HRPAP20 phosphorylation, suggesting that other novel or atypical PKC isoforms may be involved. Together, we suggest that HRPAP20 may be regulated by PRL and EGF in hormone-responsive breast cancer with the involvement of an interaction between HRPAP20, CaM and PKC. Finally, we show that siRNA knockdown of HRPAP20 expression in tumor cells resulted in a markedly diminished rate of tumor/xenograft growth. From these results, we propose that HRPAP20 expression may be required for tumor progression in breast cancer. Knowledge of the role of HRPAP20 in growth factor and hormone-mediated pathways would provide additional information regarding its potential as a chemotherapeutic target for breast cancer.
Dr. Arthur Buckley (Advisor)
173 p.
HRPAP20; Calmodulin; PKC; Invasion; Breast Cancer

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Citations

  • SHUKLA, M. N. (2007). HRPAP20: A NOVEL CALMODULIN-BINDING PHOSPHOPROTEIN INVOLVED IN TUMOR PROGRESSION [Doctoral dissertation, University of Cincinnati]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1175629479

    APA Style (7th edition)

  • SHUKLA, MANASI. HRPAP20: A NOVEL CALMODULIN-BINDING PHOSPHOPROTEIN INVOLVED IN TUMOR PROGRESSION. 2007. University of Cincinnati, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ucin1175629479.

    MLA Style (8th edition)

  • SHUKLA, MANASI. "HRPAP20: A NOVEL CALMODULIN-BINDING PHOSPHOPROTEIN INVOLVED IN TUMOR PROGRESSION." Doctoral dissertation, University of Cincinnati, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1175629479

    Chicago Manual of Style (17th edition)

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© 2007, all rights reserved.
This open access ETD is published by University of Cincinnati and OhioLINK.