The SH2D5 signaling molecule is a previously uncharacterized adaptor-like
protein, containing an N-terminal phosphotyrosine binding (PTB) domain and a noncanonical
Src Homology-2 (SH2) domain. With an antibody that I developed, I have
shown that SH2D5 is highly enriched throughout adult brain regions. Furthermore,
SH2D5 is localized to purkinjie cells in the cerebellum, the cornu ammonis (CA) of
the hippocampus and pyramidal cells in the cortex. Despite harbouring two potential
phosphotyrosine (pTyr) recognition domains, SH2D5 binds minimally to pTyr
ligands. To discover the interaction partners of SH2D5 I conducted an
immunoprecipitation/ mass spectrometry (IP/MS) screen from cultured Human
Embryonic Kidney (HEK) 293T and Neuro2A cells along with murine brain lysates.
These experiments revealed novel binding partners to SH2D5 including a prominent
association with the RacGAP protein, Breakpoint Cluster Region protein (BCR),
which is also highly expressed in brain. I have defined the interaction between SH2D5
and BCR and show that the PTB domain of SH2D5 engages an NxxF motif located
within the N-terminal region of BCR. To address the biological significance of
SH2D5, I utilized an siRNA approach to deplete the neuroblastoma cell-line, B35, of
iii
SH2D5. In these assays, B35 cells display a cell rounding phenotype and grow in a
lattice formation. Furthermore, upon SH2D5 depletion these cells display low levels
of activated Rac, associated with cell rounding. Taken together, these data reveal the
first characterization of the SH2D5 signaling protein, its novel interaction with BCR
and phenotype in neuronal-like cells. These data signify a biological function for
SH2D5 in neurobiologic signaling perhaps applicable to learning and memory.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/32723 |
| Date | 21 August 2012 |
| Creators | Gray, Elizabeth Jean |
| Contributors | Pawson, Tony |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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