Date of Award

5-2008

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Molecular Sciences

Research Advisor

Ramareddy V. Guntaka, Ph.D.

Committee

John Cox, Ph.D. George Hilliard, Ph.D. Rajendra Raghow, Ph.D. Pat Ryan, Ph.D.

Abstract

Y-box binding protein, YB-1, is a member of the cold shock domain superfamily of proteins. It is involved in a plethora of cellular functions, including cell proliferation. The molecular mechanisms governing the involvement of YB-1 in cell proliferation are still unclear. Earlier studies done in chicken pre-B lymphocyte DT-40 cells in our laboratory have shown that a targeted disruption in one allele of chicken YB-1

(Chk-Yb-1b) gene at its N-terminal domain resulted in multiple abnormalities in the heterozygous mutants, including slower growth rate, abnormal cell morphology, increased cell size, increased genomic DNA content and significant changes in levels of cell cycle specific genes like the cyclin dependent kinase inhibitor - p21.

The current study is based on the hypothesis that the defects seen in the heterozygous mutant DT-40 cells were due to a dominant negative effect of the potential YB-1 N-terminal truncated protein. The goal of this study is to determine if the introduction of the N-terminal region of YB-1 mimics the defects seen in the mutant DT-40 cells and to identify cell cycle specific proteins that interact with YB-1.

Using rat hepatoma cells (H411E) as a model system, we introduced three different YB-1 N-terminal fusion proteins, i) APYB77GFP, the 77 amino acid long full length YB-1 N-terminal sequence containing clone, ii) APYB36GFP, the 36 amino acid long peptide with the internal deletion Δ12-52 (APYB36Δ12-52GFP represented as APYB36GFP) and iii) APYB26GFP, the 26 amino acid proline rich YB-1 N-terminal sequence containing clone and investigated their effects on incubated cells. Our findings show that introduction of the APYB26GFP and APYB77GFP proteins resulted in cell cycle arrest at the G2/M phase. In contrast, incubation with the APYB36GFP protein showed a lower proportion of G2/M phase cells. APYB26GFP and APYB77GFP protein incubations also resulted in significant apoptosis, which was confirmed by Annexin V staining, DNA fragmentation analysis and nuclear breakdown analysis. The APYB36GFP incubation showed lesser percentage of apoptotic cells. We conclude that the YB-1 amino-end sequence plays an important role in cell proliferation and apoptosis.

We also demonstrate that YB-1 interacts with the cell cycle protein - cyclin D1, predominantly in the cytoplasm of G2/M phase blocked cells and the YB-1 N-terminus is involved in this interaction. We propose that the proline alanine rich PPAAPPAAPALSAADTK sequence present in APYB26GFP and APYB77GFP, but not in APYB36GFP is involved in this interaction, as cyclin D1 did not immunoprecipitate with APYB36GFP. We conclude that the YB-1 N-terminus interacts with Cyclin D1 in the cytoplasm of G2/M phase cells and this interaction probably sequesters the cyclin D1 in the cytoplasm leading to cell cycle arrest and apoptosis.

In summary, this study demonstrates that the amino-end domain of YB-1 plays a role in cell proliferation and apoptosis probably by sequestering cyclin D1 in the cell cytoplasm. It is likely that this process is mediated through the proline alanine rich sequence PPAAPPAAPALSAADTK, present in the N-terminus of YB-1.

DOI

10.21007/etd.cghs.2008.0161

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