Semester

Fall

Date of Graduation

1999

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Microbiology, Immunology, and Cell Biology

Committee Chair

Daniel Flynn.

Abstract

The actin filament associated protein of 110 kDa (AFAP-110) is a SH2/SH3 binding partner for Src. It has two SH2 binding motifs, one SH3 binding motif, one proline rich motif, two PH domains and one serine/threonine substrate region at its amino terminus and has been hypothesized to function as an adapter protein that mediate the effects of different signal proteins upon actin filaments. In this report, we demonstrated: (1) AFAP-110 has the ability to self-associate through carboxy terminal interactions. Analysis of the carboxy terminus of AFAP-110 reveals a leucine zipper motif. Expression of carboxy terminus as a fusion protein (GST-cterm) can affinity absorb AFAP-110 from cell lysates, and the integrity of leucine zipper motif in GST-cterm is required for the affinity absorption. FPLC confirm AFAP-110 exists in multimeric forms from monomer to tetramer in vivo; (2) Src527F transformation changes the profile of AFAP-110's self-association in vivo and abrogates the affinity absorption of AFAP-110 by GST-cterm in vitro, which is independent of tyrosine phosphorylation; (3) AFAP-110 directly binds to actin filaments through its carboxy terminus. Analysis of the carboxy terminus of AFAP-110 reveals that there are two homologies with consensus actin binding domains (ABD-1 and ABD-3) between amino acids 593-637; (4) AFAP-110 has an intrinsic ability to modulate the integrity of actin filaments and to induce the formation of lamellipodia and changes in cell shape, which may be regulated by the leucine zipper motif. Taken together, these results indicate that AFAP-110 may position itself upon actin filaments through the carboxy terminal region, and the leucine zipper motif may play a regulatory role in affecting the actin filament integrity and the formation of lamellipodia in response to different cellular signals that include Src.;In addition, we mapped two monoclonal antibody reactive epitopes within AFAP-110. Mab 4C3 recognized an epitope within the SH3 binding motif of avian AFAP-110 and did not efficiently react with mammalian homologoes of AFAP-110. Mab anti-AFAP-110 recognized an epitope within the carboxy terminus of AFAP-110, which is conserved across the species. These two monoclonal antibodies provided useful tools to study AFAP-110.

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