R01 HL51456 - Hava Avraham - New England Deaconess Hospital, Boston, MA
One of the key manifestations in HIV infection is thrombocytopenia, which was described even prior to the discovery of HIV. This researcher is investigating the role of various gene products in regulating megakaryocytopoiesis and the effect of HIV infection on megakaryocytic cells. One such gene product, termed megakaryocyte-associated tyrosine kinase, will be studied with respect to its expression during HIV infection. Initial results have indicated an inhibitory effect of HIV on expression of these genes. The results of these studies will elucidate one mechanism for retroviral impairment of megakaryocyte growth and maturation.
This grant is being supported from 1994 to 1999.
Thrombocytopenia is often associated with HIV infection. This investigator is studying the regulation of proliferation and differentiation of megakaryocytes which produce platelets. The megakaryocyte associated tyrosine kinase (MATK or CHK) is a recently identified enzyme found predominantly in human brain and hematopoietic tissues. This protein has now been isolated and its structure determined. An interesting observation of this study is that in cells stimulated with growth factors e.g., c-kit, the kinase becomes associated with the receptor of the growth factor. This observation may provide insight into the process of megakaryocyte differentiation by growth factors and production of platelets.
R01 HL44851 - David Scadden - New England Deaconess Hospital, Boston, MA
The purpose of this grant is to investigate mechanisms for dysregulated hematopoiesis following HIV infection. It is: (1) evaluating progenitor cell and bone marrow stromal cell infection in vitro by using PCR analysis of primary patient samples; (2) infecting normal primary stromal cells and immortalized human bone marrow stromal cell lines, BL, with HIV in vitro; (3) defining the functional consequences of HIV infection of marrow stromal cells, as measured by (a) the support of hematopoiesis in long-term bone marrow culture, (b) cytokine expression, (c) extracellular matrix expression and (d) cell-surface adhesion molecule expression; (4) exploring the biochemical basis for abnormalities in expression of cellular gene products induced by HIV infection of stroma; and (5) verifying the role of the altered host gene product in hematopoiesis.
This grant is being supported from 1991 to 2001.
After the initial studies did not demonstrate a clearly defined mechanism by which HIV may affect blood cell development, the principal investigator will now focus on defining hematopoiesis under uninfected conditions as a prerequisite to evaluating the role for HIV. In doing so they have defined both novel systems and novel genes potentially regulating those systems. These tools should allow greater depth in addressing the perturbations of hematopoiesis that HIV induces. The work has provided the groundwork for evaluating gene therapy strategies for HIV disease.
R01 HL55445 - Shalom Avraham - New England Deaconess Hospital, Boston, MA
An improved understanding of the regulation of blood cell growth, maturation, and function in normal and diseased states may be derived by studying signal transduction pathways. We have recently discovered in human megakaryocytes, T-cells and macrophages a novel cytoplasmic tyrosine termed RAFTK (Related Adhesion Focal Tyrosine Kinase). RAFTK is a member of the focal adhesion kinase (FAK) gene family, a signal transduction molecule that acts as a point of convergence in pathways of the action of certain integrins and cytokines. We have cloned and characterized the human and murine RAFTK cDNAs, which share 95 percent homology, indicating that RAFTK is highly conserved between these species. The RAFTK cDNA, encoding a polypeptide of 1009 amino acids, has closest homology (48 percent, 65 percent similarity) to FAK. In fetal tissues, RAFTK expression was observed only in brain while it was less restricted in adult tissues, indicating that RAFTK expression is developmentally upregulated. RAFTK was observed in human CD34+ marrow cells, primary bone marrow megakaryocytes, peripheral blood T-cells, monocyte-macrophages, and platelets. The RAFTK protein was rapidly phosphorylated on tyrosine residues in response to integrin-mediated cell adhesion and to various stimuli that enhanced intracellular calcium concentration as well as protein kinase C activation (e.g., thrombin, SCF, T-cell antigen ligation, M-CSF, collagen, and calcium ionophore A23187). Moreover, HIV infection of T-cells significantly reduced RAFTK activation after T-cell receptor ligation or lectin stimulation. This suggests a new area of study in the pathogenesis of T-cell dysfunction in AIDS.
We aim to further characterize this novel signaling molecule with regard to its role in normal and HIV infected cells. Specifically we propose to: a) purify and characterize RAFTK; b) study the role of RAFTK protein in normal blood cell signal transduction and after HIV infection of susceptible cells c) map the functional domains of RAFTK and determine the structure-function relationships of RAFTK; and d) determine if RAFTK expression is required for normal hematopoietic proliferation and differentiation. These studies should provide insights into the biology of normal and HIV-perturbed blood cell growth and function.
This grant is being supported from 1996 to 2001.
RAFTK is tyrosine phosphorylated and localized in CMK megakaryocytic cell lines, activated with SCF or PMA, in focal adhesion like structures. The phosphorylation and activation of RAFTK may be integrin mediated and depends on the nature of the cell under study. The current data suggest that this kinase is involved in signal transduction in hematopoietic cells.