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Glycogen synthase kinase-3 (GSK-3) is a serine/threonine protein kinase which plays a key role in several signaling pathways and its homologues have been identified in most eukaryotes. Since GSK3£]is an essential protein kinase that regulates numerous functions within the cell, an effort to survey possible GSK3£]- interacting proteins from a human testis cDNA library using the yeast two-hybrid system is made. Two interesting candidates are chosen to characterize their functions in this study. One is a centrosomal protein, hNinein, and the other is a novel inhibitor of GSK3£], designated as GSKIP (GSK3£] interaction protein). In the first part of the present thesis we describe the identification of four diverse CCII-termini of human hNinein isoforms, including a novel isoform 6, by differential expression in a tissue-specific manner. In a kinase assay, the CCII region of hNinein isoforms provides a differential phosphorylation site by GSK3£]. In addition, either N-terminal or CCIIZ domain disruption may cause hNinein conformational change which recruits £^-tubulin to centrosomal or non-centrosomal hNinein-containing sites. Further, depletion of all hNinein isoforms caused a significant decrease in the £^-tubulin signal in the centrosome. In domain swapping, it clearly shows that the CCIIX-CCIIY region provides docking sites for £^-tubulin. Moreover, nucleation of microtubules from the centrosome is significantly affected by the overexpression of either the full-length hNinein or CCIIX-CCIIY region. Taken together, these results show that the centrosomal targeting signals of hNinein have a role not only in regulating hNinein conformation, resulting in localization change, but also provide docking sites to recruit £^-tubulin at centrosomal and non-centrosomal sites. In the second part of the thesis we describe another candidate, GSK3£]interaction protein (GSKIP), to characterize its functions in neuron differentiation. We use human neuroblastoma SH-SY5Y cells as a model of neuronal cell differentiation. When overexpression of GSKIP prevents neurite outgrowth from RA-mediated differentiation, this result is similar to the presence of LiCl or SB415286, an inhibitor of GSK3£]. Further, GSKIP regulates the activity of GSK3£] through protein-protein interactions rather than post-modulation and GSKIP may affect GSK3£] on neurite outgrowth via inhibiting the specific phosphorylation site of tau. In addition to inhibition of neurite outgrowth, GSKIP overexpressed in SH-SY5Y cells also promotes cell cycle progression by analyzing cell proliferation with cell growth and MTT assay. Furthermore, GSKIP raises the level of £]-catenin and cyclin D1 through inhibition of GSK3£] activity in RA-mediated differentiation SH-SY5Y cells. Taken together, the data suggest that GSKIP, a dual functional molecule, is able to inhibit neurite outgrowth and promote cell proliferation via negative regulation of GSK3£] activity in RA-mediated differentiation of SH-SY5Y cells.

LIM kinase 1 (LIMK1) is a unique dual specificity serine/threonine kinase containing two N-terminal LIM domains in tandem, a PDZ domain and a C-terminal catalytic domain. LIMK1 is involved in modulation of actin cytoskeleton through inactivating phosphorylation of the ADF (actin depolymerization factor) family protein cofilin. Recent studies have shown that LIMK1 is upregulated in breast and prostate cancer cells and tissues, promotes metastasis in animals and induces acquisition of an invasive phenotype when ectopically expressed in benign prostate epithelial (BPH) cells. Furthermore, overexpression of LIMK1 was associated with altered sub cellular localization of the membrane type 1 matrix metalloprotease (MT1-MMP). Matrix metalloproteases (MMPs) are a family of zinc dependant proteolytic enzymes that hydrolyze extra cellular matrix and cell surface molecules. A number of MMPs including MMP-2, MMP-9 and their activator MT1-MMP are over expressed in a variety of cancers including prostate cancer. The abundant expression of these enzymes contributes to changes in the tumor microenvironment, which facilitate degradation of the surrounding collagen matrix and migration of cells through the matrix defects. In this study, we show that MMPs are involved in LIMK1 induced invasion of otherwise non-invasive BPH cells. We also show that (a) the kinase activity of LIMK is not essential for the invasive behavior of the cells and (b) the absence of LIM domains significantly retards cell invasion. We have established transfected sub lines of BPH cells stably expressing 1) constitutively active LIMK1 (BPHLCA), 2) kinase dead LIMK1 (BPHLKD) and 3) only the kinase domain of LIMK1 (BPHLK) for our study. In vitro invasion assays revealed that LIMK1 induced invasion was inhibited by the MMP specific inhibitor, GM6001, and that cells expressing kinase-dead LIMK1 were equally invasive. Furthermore, BPH cells expressing LIMK1 mutants expressed higher amounts of MMP-2 and MMP-9. Substrate zymography revealed increased concentration of secreted MMP-2 and MMP-9 in the media of BPHLCA and BPHLK cells respectively compared to BPHV (vector control) cells. Quantitative RT-PCR also showed a ~10 fold increase in the steady state concentration of MMP-2 in BPHLCA cells compared to the control BPHLV cells. Expression of active LIMK1 stimulated cell-surface expression of MT1-MMP in BPHLCA cells as determined by flow cytometry. A modest increase in expression of MT1-MMP was noted in BPHLKD cells compared to BPHLK and BPHV cells. Immunoflourescence analysis indicated differential localization of MT1-MMP and LIMK1 in BPH cells expressing different mutants of LIMK1. Co-localization of LIMK1 and MT1-MMP in the plasma membrane and in the perinuclear region was also evident in these cells. Furthermore, here we provide evidence that suggests a functional role for phosphorylated (activated) LIMK1/2 (p-LIMK1/2) during mitosis through its association with γ-tubulin. Immunoflourescence analysis showed distinct co-localization of γ -tubulin and p-LIMK1/2 in the centrosomes during mitosis from early prophase to the beginning of telophase. No association was seen in the interphase or in late telophase. Phospho-LIMK1/2 was co-precipitated in immunoprecipitates of γ -tubulin using an anti- γ -tubulin antibody suggesting a physical association between these proteins in a complex. This finding reveals a novel role of LIMK1 in the mitotic process. In summary, our data suggests that MMPs are involved in LIMK1 induced invasion of prostate epithelial cells, and that this effect is mediated through altered expression and activation of specific MMPs. Furthermore, LIMK1 induced invasion is dependant on the presence of LIM domains more than the kinase activity. Finally, we show that phosphorylated LIMK1 and LIMK2 are involved in the mitotic process in a stage specific manner through its association with the centrosomal protein γ -tubulin. Because LIMK1 promotes invasion in vitro, regulates expression of MMPs, and is involved in mitotic processes, it is an attractive drug target for prostate cancer therapy.

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