Protein-tyrosine kinases have been broadly implicated in the control of both normal and neoplastic cell proliferation. For example, the binding of many polypeptide growth factors to their cellular receptors results in the rapid activation of a receptor- associated protein-tyrosine kinase that phosphorylates the receptor itself, as well as a number of cellular proteins. Furthermore, the ability of many oncogenic retroviruses to transform cells in dependent on their encoding and expressing a constitutively active protein-tyrosine kinase. As an initial step towards our ultimate goal of understanding the mechanisms whereby tyrosine phosphorylation regulates cell growth, we recently produced a monoclonal antibody that has high affinity and great specificity for phosphotyrosyl-proteins. Making extensive use of this antibody, the proposed studies seek to identify cellular functions related to cell proliferation that are controlled by protein- tyrosine kinases and their cellular substrates. These studies seek to : (i) identify cellular responses to growth factors and to oncogenic proteins that are perturbed by antibodies to phosphotyrosine; (ii) identify cellular functions influenced by individual phosphotyrosyl-proteins by inserting them into quiescent, non-transformed cells; (iii) prepare antibodies to three cellular phosphotyrosyl-proteins that appear to be involved in tyrosine-kinase responses and to use the antibodies to examine the expression, distribution and subcellular location of these three proteins as well as to examine and perturb their functions in intact cells. A main approach in all these aims involves the insertion of antibodies (or phosphotyrosyl proteins) into cells by osmotic lysis of pinocytic vesicles.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA039235-05
Application #
3178042
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1984-08-01
Project End
1991-03-31
Budget Start
1989-04-01
Budget End
1990-03-31
Support Year
5
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Roger Williams Hospital
Department
Type
DUNS #
City
Providence
State
RI
Country
United States
Zip Code
02908
Cheng, J C; Frackelton Jr, A R; Bearer, E L et al. (1995) Changes in tyrosine-phosphorylated p190 and its association with p120 type I and p100 type II rasGAPs during myelomonocytic differentiation of human leukemic cells. Cell Growth Differ 6:139-48
Frackelton Jr, A R; Kumar, P S; Kannan, B et al. (1993) Tyrosine phosphorylated proteins in chronic myelogenous leukemia. Leuk Lymphoma 11 Suppl 1:125-9
Buzzi, M; Lu, L; Lombardi Jr, A J et al. (1992) Differentiation-induced changes in protein-tyrosine phosphatase activity and commensurate expression of CD45 in human leukemia cell lines. Cancer Res 52:4027-35
Beitz, J G; Kim, I S; Calabresi, P et al. (1991) Human microvascular endothelial cells express receptors for platelet-derived growth factor. Proc Natl Acad Sci U S A 88:2021-5
Frackelton Jr, A R; Posner, M; Kannan, B et al. (1991) Generation of monoclonal antibodies against phosphotyrosine and their use for affinity purification of phosphotyrosine-containing proteins. Methods Enzymol 201:79-92
Huhn, R D; Cicione, M E; Frackelton Jr, A R (1989) Identification of tyrosine-phosphorylated colony-stimulating factor 1 (CSF-1) receptor and a 56-kilodalton protein phosphorylated in intact human cells in response to CSF-1. J Cell Biochem 39:129-37
Sengupta, A; Liu, W K; Yeung, Y G et al. (1988) Identification and subcellular localization of proteins that are rapidly phosphorylated in tyrosine in response to colony-stimulating factor 1. Proc Natl Acad Sci U S A 85:8062-6
Huhn, R D; Posner, M R; Rayter, S I et al. (1987) Cell lines and peripheral blood leukocytes derived from individuals with chronic myelogenous leukemia display virtually identical proteins phosphorylated on tyrosine residues. Proc Natl Acad Sci U S A 84:4408-12
Bell, J C; Mahadevan, L C; Colledge, W H et al. (1987) Abelson-transformed fibroblasts contain nuclear phosphotyrosyl-proteins which preferentially bind to murine DNA. Nature 325:552-4
Daniel, T O; Tremble, P M; Frackelton Jr, A R et al. (1985) Purification of the platelet-derived growth factor receptor by using an anti-phosphotyrosine antibody. Proc Natl Acad Sci U S A 82:2684-7