University at Buffalo
521 Biomedical Research Building
Buffalo, NY 14214
PH: (716) 829-3491
Web: http://deptdirectory.med.buffalo.edu/pro...
E: aedelman@buffalo.edu
530 Biomedical Research Building
Buffalo, NY 14214
PH: (716) 829-3057
Research
Signal transduction; protein phosphorylation; enzymology; proteomics; molecular biology
Lissencephaly
Protein kinase catalyzed protein phosphorylation is well established as playing a major role in the function of the fully differentiated nervous systems of higher metazoan organisms. Recently, the role of protein kinases in nervous system development has now also become an area of very active investigation. A new family of neural-specific protein kinases comprised of doublecortin kinases (DCKs) 1 and 2 has recently been described. These kinases are related in amino acid sequence identity to the protein doublecortin (DCX). DCX was first identified as the gene mutated in X-lissencephaly and doublecortex syndrome, a severe malformation of the cerebral cortex occurring during fetal development, and manifested clinically as epilepsy and mental retardation. The role of the encoded protein DCX, in embryonic cortical development has been attributed to its mediation of neuroblast migration, and this has been linked at the molecular level to its ability to polymerize and stabilize microtubules (MTs). The DCKs (via their DC domains) share with DCX the ability to modulate MT dynamics. There are at present, however, no data to suggest what role the DCK kinase domains may play in neuronal cytoskeletal dynamics nor is there information as to the upstream signaling pathways which regulate DCK activity. Yet there is compelling biochemical and genetic evidence indicating that the respective kinase activities of DCKs are essential for their function. As part of a broader line of investigation into the role of phosphorylation in the regulation and function of the DCKs the project described here is designed to accomplish the following Specific Aims:
- Specific Aim 1: To identify the molecular components of a DCK activator
- Specific Aim 2: To elucidate the mechanism of DCK activation
Neuroblastoma
Dysregulation of gene expression, through a variety of different mechanisms is a hallmark of cancer. Current concepts of the intracellular mechanisms which cause or contribute to the initiation or maintenance of the malignantly transformed state have emphasized growth factor-dependent signaling pathways. In this proposal we ask whether an alternate signaling pathway, one which is triggered by intracellular calcium (Ca2+i) and which regulates gene expression may play a role in certain types of cancer. Calmodulin (CaM) kinase IV (CaMKIV), is a predominantly nuclear protein kinase which is activated allosterically by Ca2+/CaM, and by phosphorylation catalyzed by CaM kinase kinases (CaMKKs). The CaMKK/CaMKIV module (CaMKIV cascade) serves to transduce stimuli-dependent alterations in Ca2+i into transcriptional regulation of a broad array of sequence-specific transcription factors and co-regulators. We propose to examine the roles of the CaMKIV cascade in proliferation, survival and oncogenesis in , human neuroblastoma BE(2)C cells in vitro and in vivo. As neuroblastoma is the most commonly diagnosed cancer of children elucidation of intracellular mechanisms by which it propagates and spreads, is important for the design of new therapeutic interventions for this often deadly malignancy. The Specific Aims are:
Specific Aim 1:
- To dissect the respective roles of the components of the CaM kinase IV cascade in proliferation and survival of human neuroblastoma cells;
- To define the involvement of the CaMKKs in the CaM kinase IV cascade in BE(2)C cells;
- To determine the timing and mechanism of cell cycle arrest caused by inhibition of the CaM kinase IV cascade in BE(2)C cells;
- To assess whether exit from the cell cycle caused by inhibition of the CaM kinase IV cascade is associated with apoptosis or differentiation of BE(2)C.
Specific Aim 2: To identify and define the cellular roles of targets of the CaM kinase IV cascade in human neuroblastoma cells.
Specific Aim 3: To determine the potential for oncogenicity of the CaM kinase IV cascade in cultured cells and in tumors in vivo
- To determine whether forced expression of CaM kinase IV cascade components induces a transformed phenotype in non-transformed cells; and conversely, whether their inhibition suppresses the transformed phenotype in cancer cells;
- To determine if inhibition of the CaM kinase IV cascade affects growth and angiogenesis of neuroblastoma tumors in athymic (nude) mice.
Prostate Cancer
Protein kinases are important intracellular regulators of cell proliferation and survival and are promising molecular targets for the development of new anti-cancer therapies. The Ca2+-calmodulin (CaM)-dependent protein kinase cascade is comprised of an upstream CaM kinase kinase, CaMKKα or β(CaMKK1, CaMKK2 respectively), which phosphorylates and activates a downstream CaM kinase such as the nuclear transcriptional regulator, CaMKIV. Expression profiling studies identified CaMKKβ as an androgen-dependent gene and as up-regulated in prostate cancer. We have obtained preliminary data that CaMKKα, CaMKKβ and CaMKIV are up-regulated in prostate tumors from a transgenic mouse model of prostate cancer (TRAMP) and that the selective CaMKK inhibitor, STO-609 reduces proliferation and viability of prostate cancer cell lines.
The central hypothesis of this proposal is that the CaM kinase cascade plays a role in the pathogenesis of prostate cancer and represents a molecular target for its therapeutic intervention. This hypothesis will be tested by two Specific Aims:
- Specific Aim 1: To evaluate the oncogenic potential of the CaM kinase cascade in prostatic cells and in transplanted mouse models of prostate cancer.
- Specific Aim 2: To determine the efficacy of targeted inhibition of the CaM kinase cascade on tumor progression in the TRAMP mouse model of prostate cancer.
GCG (Genetics Computer Group) suite of programs
- mouse
- rat
- E.coli
- Yeast
- rabbit- for specific applications
- Ca2+/calmodulin dependent protein kinases
- Doublecortin
- doublecortin kinases
Lissencephaly (smooth brain), an X-linked neurological disorder; Neuroblastoma; Prostate Cancer
- RNA interference
- protein kinase characterization
- MALDI
- mass spectrometry
- standard molecular biology techniques
- Quantitative PCR
- Immunocytochemistry
For people interested in making their own antibodies, I would be able to advise. We routinely make our own antibodies when commercial antibodies are not available or not adequate.
- BE(2)C neuroblastoma
- HEK293
- Prostate cancer cell lines: PC3, LNCaP, TRAMP-C2
Lissencephaly (smooth brain); Neuroblastoma; Prostate Cancer
Brain, prostate
Hawley, S.A., Pan, D.A., Mustard, K.J. Ross, L. Bain, J., Edelman, A.M., Frenguelli, B.G. and Hardie, D. G.; Calmodulin-dependent protein kinase kinase-%u03B2 is an alternative upstream kinase for AMP-activated protein kinase; Cell Metabolism; 2005 Jul; 2(1); 9-19
Edelman AM, Kim WY, Higgins D, Goldstein EG, Oberdoerster M, Sigurdson W; Doublecortin kinase-2: A novel doublecortin-related protein kinase associated with terminal segments of axons and dendrites.; Journal of Biological Chemistry; 2005 Mar; 280(9); 8531-8543
Qin, H., Raught, B., Sonenberg, N., Goldstein E.G. and Edelman, A.M.; Phosphorylation screening identifies translational initiation factor 4GII as an intracellular target of Ca2 /calmodulin-dependent protein kinase I; J. Biol. Chem.; 2003 Sep
Shang, L., Kwon, Y-G, Nandy, S., Lawrence, D.S. and Edelman, A.M.; Catalytic and regulatory domains of doublecortin kinase-1; Biochemistry; 2003; 42; 218-2194