Presentation Title

Biochemical Characterization and Developmental Expression Profiling of the Predicted CRK/CRKL-SH2 Binding Partner WBP2

Presenter's Name(s)

Brigitte N. DurieuxFollow

Abstract

Chicken tumor virus 10 regulator of kinase (CRK) and CRK-like (CRKL) are two members of a family of adaptor (regulatory) proteins which facilitate the association of proteins into complexes during fundamental cellular processes, including cell proliferation, cell adhesion, phagocytosis, apoptosis, the regulation of gene expression, and neuronal migration. They have near-identical structures; each possesses a Src homology 2 (SH2) domain, and two Src homology 3 (SH3) domains. These are protein-protein interaction domains which recognize specific motifs in other proteins. This SH2-SH3-SH3 structure allows CRK and CRKL to proximally associate binding partners. Proper development of several brain structures require migrating neurons to position themselves in a specific three-dimensional layering pattern, directed by surrounding biochemical signals. CRK and CRKL are essential for this process of neuronal migration during development. As these proteins are adaptors, the next “steps” of that cell migration pathway could be linked to CRK and CRKL binding partners.

A recent study has identified several potential CRK and CRKL binding partners using novel in silico methods; WBP2 and PDLim5 are among these. As mutations in the WBP2 gene have been linked to autosomal recessive deafness (non-syndromic sensorineural hearing loss), which involves non-functionality of inner ear neural receptors, the auditory nerve and its downstream synapses, and PDLim5 is a protein known to contribute to the regulation of dendritic spine morphogenesis in neurons, these proteins are high-priority putative CRK and CRKL binding candidates. In this study, we biochemically validated interactions between the CRKL-SH2 domain and WBP2 and PDLim5, likely in a phosphotyrosine-dependent manner. This was done via a pulldown assay with a CRKL-SH2 domain fused to a glutathione resin, and Western Blotting for visualization. In addition, we performed an in situ experiment with zebrafish (Danio rerio), and found WBP2 to be actively transcribed in the brain during embryonic development. Further work will investigate the interaction and phosphorylation-dependence, of the interaction between WBP2 and the CRKL-SH2 domain using Mass Spectrometry.

Primary Faculty Mentor Name

Dr. Bryan Ballif

Secondary Mentor Name

Dr. Alicia Ebert

Graduate Student Mentors

Anna Schmoker, Caroline Dumas, Helaina Stergas

Status

Undergraduate

Student College

College of Arts and Sciences

Program/Major

Biochemistry

Primary Research Category

Biological Sciences

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Biochemical Characterization and Developmental Expression Profiling of the Predicted CRK/CRKL-SH2 Binding Partner WBP2

Chicken tumor virus 10 regulator of kinase (CRK) and CRK-like (CRKL) are two members of a family of adaptor (regulatory) proteins which facilitate the association of proteins into complexes during fundamental cellular processes, including cell proliferation, cell adhesion, phagocytosis, apoptosis, the regulation of gene expression, and neuronal migration. They have near-identical structures; each possesses a Src homology 2 (SH2) domain, and two Src homology 3 (SH3) domains. These are protein-protein interaction domains which recognize specific motifs in other proteins. This SH2-SH3-SH3 structure allows CRK and CRKL to proximally associate binding partners. Proper development of several brain structures require migrating neurons to position themselves in a specific three-dimensional layering pattern, directed by surrounding biochemical signals. CRK and CRKL are essential for this process of neuronal migration during development. As these proteins are adaptors, the next “steps” of that cell migration pathway could be linked to CRK and CRKL binding partners.

A recent study has identified several potential CRK and CRKL binding partners using novel in silico methods; WBP2 and PDLim5 are among these. As mutations in the WBP2 gene have been linked to autosomal recessive deafness (non-syndromic sensorineural hearing loss), which involves non-functionality of inner ear neural receptors, the auditory nerve and its downstream synapses, and PDLim5 is a protein known to contribute to the regulation of dendritic spine morphogenesis in neurons, these proteins are high-priority putative CRK and CRKL binding candidates. In this study, we biochemically validated interactions between the CRKL-SH2 domain and WBP2 and PDLim5, likely in a phosphotyrosine-dependent manner. This was done via a pulldown assay with a CRKL-SH2 domain fused to a glutathione resin, and Western Blotting for visualization. In addition, we performed an in situ experiment with zebrafish (Danio rerio), and found WBP2 to be actively transcribed in the brain during embryonic development. Further work will investigate the interaction and phosphorylation-dependence, of the interaction between WBP2 and the CRKL-SH2 domain using Mass Spectrometry.