UVM Theses and Dissertations
Format:
Print
Author:
Cheerathodi, Mujeeburahiman
Dept./Program:
Biology
Year:
2012
Degree:
PhD
Abstract:
This work describes the proteomic identification of CrkL-SH3 binding proteins from embryonic brain and liver. Several of these proteins were individually characterized and their interaction with the CrkL-SH3 domain was validated. The implications these protein complexes have in Crk-and CrkL-dependent signal transduction, particularly Reelin signaling during mammalian brain development, are discussed. Additionally, quantitative proteomic approaches were used to identify tissue-specific CrkL-SH3 binding protein variants.
Reelin is a secreted glycoprotein which plays a crucial role in the positioning of neurons during mammalian brain development. Mutation in the Reelin gene leads to an ataxic phenotype called Reeler. Reelin binds to the receptors VLDLR and ApoER2 leading to the activation of the Src family of tyrosine kinases (SFK) and the subsequent phosphorylation of the adaptor protein Disabled-I (Dabl). Crk (CTIO regulator of kinases) and CrkL (Crk-Like) are adaptor proteins comprised of one SH2 domain and two SH3 domains which are recruited to phosphorylated-Dab I in a Reelin-dependant manner. Using their SH3 domains Crk and CrkL can recruit effector proteins involved in Reelin signaling. Biochemical and molecular studies have identified 21 Crk-or CrkL-SH3 interacting proteins, including C3G, a guanine nucleotide exchange factor for Rapl. C3G is recruited to the Reelin signaling complex and is activated in response to Reelin.
Mice homozygous for a hypomorphic allele of C3G do not fully phenocopy compound loss of Crk and CrkL suggesting additional Crk/L-SH3 binding effector proteins are recruited to the Reelin signaling complex. As a first step to test this we conducted a large-scale identification of CrkL-SH3 binding proteins from murine embryonic brain using affinity chromatography coupled with tandem mass spectrometry. A total of 101 CrkL-SH3 binding proteins were identified including 86 novel ones. Gene ontology analysis found these proteins to be enriched the following functional categories: cell adhesion, cell structure and mobility, and signal transduction. Using biochemical studies, we identified Lipoma Preferred Partner homologue (LPP), a zyxin family protein associated with focal adhesions and cell-cell contacts, as a potential effector in Reelin signaling.
Given our analysis did not identify some known Crk/L-SH3 binding proteins we hypothesized this was due to developmental stage-and tissue-specific differences. As a first step to compare CrkL-SH3 binding proteins from different tissues we used a quantitative proteomic approach to compare CrkL-SH3 binding proteins from embryonic murine brain and liver. Not only were tissue-specific protein quantifications observed but we also detected important differences in the relative abundances of tissue-specific molecular weight-based protein variants. In short, this study broadened the understanding of CrkL-SH3 binding proteins and highlighted the strengths, limitations and future goals of comprehensive analyses in mass-spectrometry-based proteomic approaches.
Reelin is a secreted glycoprotein which plays a crucial role in the positioning of neurons during mammalian brain development. Mutation in the Reelin gene leads to an ataxic phenotype called Reeler. Reelin binds to the receptors VLDLR and ApoER2 leading to the activation of the Src family of tyrosine kinases (SFK) and the subsequent phosphorylation of the adaptor protein Disabled-I (Dabl). Crk (CTIO regulator of kinases) and CrkL (Crk-Like) are adaptor proteins comprised of one SH2 domain and two SH3 domains which are recruited to phosphorylated-Dab I in a Reelin-dependant manner. Using their SH3 domains Crk and CrkL can recruit effector proteins involved in Reelin signaling. Biochemical and molecular studies have identified 21 Crk-or CrkL-SH3 interacting proteins, including C3G, a guanine nucleotide exchange factor for Rapl. C3G is recruited to the Reelin signaling complex and is activated in response to Reelin.
Mice homozygous for a hypomorphic allele of C3G do not fully phenocopy compound loss of Crk and CrkL suggesting additional Crk/L-SH3 binding effector proteins are recruited to the Reelin signaling complex. As a first step to test this we conducted a large-scale identification of CrkL-SH3 binding proteins from murine embryonic brain using affinity chromatography coupled with tandem mass spectrometry. A total of 101 CrkL-SH3 binding proteins were identified including 86 novel ones. Gene ontology analysis found these proteins to be enriched the following functional categories: cell adhesion, cell structure and mobility, and signal transduction. Using biochemical studies, we identified Lipoma Preferred Partner homologue (LPP), a zyxin family protein associated with focal adhesions and cell-cell contacts, as a potential effector in Reelin signaling.
Given our analysis did not identify some known Crk/L-SH3 binding proteins we hypothesized this was due to developmental stage-and tissue-specific differences. As a first step to compare CrkL-SH3 binding proteins from different tissues we used a quantitative proteomic approach to compare CrkL-SH3 binding proteins from embryonic murine brain and liver. Not only were tissue-specific protein quantifications observed but we also detected important differences in the relative abundances of tissue-specific molecular weight-based protein variants. In short, this study broadened the understanding of CrkL-SH3 binding proteins and highlighted the strengths, limitations and future goals of comprehensive analyses in mass-spectrometry-based proteomic approaches.