Ionizing radiation induces alterations in phosphotyrosyl proteins : the effects of cellular reducing capacity / Stephen William Tuttle.

Format:

Book

Manuscript

Microformat

Thesis/Dissertation

Author/Creator:

Tuttle, Stephen William.

Contributor:

Biaglow, John, advisor.

University of Pennsylvania.

Language:

English

Subjects (All):

Penn dissertations--Biochemistry.

Biochemistry--Penn dissertations.

Biochemistry.

Academic Dissertations as Topic.

Medical Subjects:

Biochemistry.

Academic Dissertations as Topic.

Local Subjects:

Penn dissertations--Biochemistry.

Biochemistry--Penn dissertations.

Physical Description:

xiii, 249 pages : illustrations ; 29 cm

Production:

1996.

Summary:

While the incidence of radiation induced apoptosis is relatively low in the wild type CHO line-K1, a substantial incidence of radiation-induced apoptosis is observed in the G6PD$\sp-$ CHO cell line, E48. This occurs in spite of the fact that there is no significant difference in the initial level of radiation-induced DNA damage between the G6PD$\sp-$ and wild type cell lines. The tyrosine kinase inhibitor genistein selectively protects the G6PD$\sp-$ cells from radiation-induced apoptosis, resulting in an increase in clonogenic survival. Therefore, we have examined radiation-induced changes in phosphotyrosyl proteins in vivo to identify the phosphotyrosyl proteins which respond to ionizing radiation.

Two proteins, migrating with relative molecular weights of 85 and 120 kDa on SDS-PAGE display a radiation dependent increase in phosphotyrosine content, while a third phosphotyrosyl protein with a relative molecular weight of 60 kDa is dephosphorylated in irradiated cells. We have identified the 60 kDa radiation-sensitive phosphotyrosyl protein as pp59$\rm\sp{fyn},$ a member of the src-family of non-receptor tyrosine kinases. pp59$\rm\sp{fyn}$ is activated in response to ionizing radiation both in whole cells and in vitro, suggesting that pp59$\rm\sp{fyn}$ may act as a radiation sensor in the cell. However the activity observed for pp59$\rm\sp{fyn}$ isolated from irradiated whole cells is significantly higher then the activity of partially purified pp59$\rm\sp{fyn}$ irradiated in vitro.

Even though pp59$\rm\sp{fyn}$ displays a radiation-dependent dephosphorylation on tyrosine residues in vivo, increasing amounts of pp59$\rm\sp{fyn}$ are observed in the anti-phosphotyrosine immunoprecipitates. The 120 kDa radiation-sensitive phosphotyrosyl protein observed in CHO cell lysates has been identified as focal adhesion kinase-pp125$\rm\sp{FAK},$ a novel non-receptor tyrosine kinase. The autophosphorylation site in pp125$\rm\sp{FAK}$ is located within a consensus pp59$\rm\sp{fyn}$ SH2 domain binding site. Radiation induced tyrosine phosphorylation of pp125$\rm\sp{FAK}$ results in the formation of a stable complex between pp125$\rm\sp{FAK}$ and pp59$\rm\sp{fyn}$ in vivo. Formation of this protein complex maintains pp59$\rm\sp{fyn}$ in its active conformation. Increased activity of pp125FAK is also observed in irradiated cells. Whether radiation-induced tyrosine phosphorylation and activation of pp125$\rm\sp{FAK}$ and pp59$\rm\sp{fyn}$ has any significance in radiation-induced apoptosis has yet to be determined.

Notes:

Supervisor: John Biaglow.

Thesis (Ph.D. in Biochemistry) -- University of Pennsylvania, 1996.

Includes bibliographical references and index.

Local Notes:

University Microfilms order no.: 97-13019.

OCLC:

187469489