OBJECTIVES OF STUDYConfirm the interaction between MAP1B and Fyn Determine whether Fyn SH2 domain phosphorylation alters MAP1B bindingHYPOTHESISMAP1B, a microtubule associated protein, and Fyn interact via Fyns SH2 domain.

EXPERIMENTAL APPROACH

Functional Investigation of the Novel Binding Partners Src family kinase Fyn and Microtubule Associated Protein MAP1BAlexandra Frank1 and Karen Hinkle11Department of Biology and Physical Education, Norwich University, Northfield VT 05663 2Department of Biology, University of Vermont, Burlington, VT 05405 CONCLUSIONS AND SIGNIFICANCEACKNOWLEDGEMENTSABSTRACT Src family kinases (SFK) are non-receptor tyrosine kinases that act as signaling mediators in many cellular processes including proliferation, differentiation, survival, adhesion, apoptosis, and motility. When abnormal cellular processes occur, SFKs have been identified to be highly expressed, resulting in cancers. Fyn, one of the 10 proteins that compose this group of kinases, has been mainly associated with immune and neurological function. Fyn presents multiple phosphorylation sites that can ultimately alter its activity. Upon identifying a series of novel proteins which bind and are potentially phosphorylated by Fyn, this study further explores the role of MAP1B. MAP1B is a protein that has been associated with tyrosination of the alpha-tubulin in neuronal microtubules. Upon phosphorylation of MAP1B it has been proposed to cause cytoskeletal changes, effecting neurite extensions. The overall goal of this study was to confirm that MAP1B binds with Fyn. This study was modeled using HEK293 cells which were transfected with MAP1B DNA, as well as cells that were taken through a mock transfection as a control. This research is of value because it can help to better understand the over-action of pathways that signal cancer cell growth, specifically the association with neuronal development that is associated with Fyn and MAP1B. With a better understanding of these pathways, it can lead to the future direction of being able to manipulate these pathways to stop cancerous cell growth through pharmaceutical means or other means.

BACKGROUND

Figure 1: Representation of the structural domain of SFK Fyn. A) Fyn is composed of three structural domains, the SH3 domain, SH2 domain, and kinase domain. This study specifically analyzes the function of the Fyns SH2 domain, focusing on its interaction with MAP1B. When SFKs are phosphorylated they are left in an open formation. This allows for novel proteins to potentially bind with the SH2 domain of Fyn. B) This figure depicts the interaction that Fyns SH2 domain and kinase domain when it is not phosphorylates. The domains of Fyn will bind with each other, leaving Fyn in a closed formation. This prevents novel proteins from binding with Fyn. C) Depicts the crystal structure of Fyn's SH2 domain. Figure adapted from B. Ballif. D) Table explaining what is known about MAP1B. RESULTS The purpose of this study was to confirm that MAP1B and Fyn bind. Understanding the interaction between MAP1B and Fyn would help us to better understand the over-action of pathways, with the hope of potentially manipulating these pathways. Funding for this work was provided by: Norwich University Summer Research Fellowship Vermont Genetics Network through Grant Number 8P20GM103449 from the INBRE Program of the National Institute of General Medical Sciences (NIGMS) and the National Center for Research Resources (NCRR), components of the National Institutes of Health (NIH). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH.

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Fyn and MAP1BInteraction StudiesTransfection of HEK293 cells with MAP1B, EGFP, mock

Cell lysis to extract proteinsProtein Assay and normalization of cell extractsFigure 2: Nanodrop of a DNA Isolation of MAP1B. In order to prepare the MAP1B DNA used in transfections, a Minikit was used to isolate the DNA. The DNA sample is then analyzed using a Nanodrop to ensure purity and to obtain the concentration. Having a low peak at 230nm and a very high peak at 260nm indicates good quality DNA. This sample had a concentration of 0.2688 ug/ul. Treatment with H202

Separation of proteins by size by SDS-PAGEDNA isolation of MAP1B plasmid

Figure 3: Coommasie Stained SDS-PAGE.To identify protein expression 90 ul samples were run through a SDS-PAGE gel Western Blot technique which separates proteins. Rather than setting this gel up for a transfer, it was stained with Coommasie Blue Stain to confirm that there was protein expression. In the first lane was the marker/ladder protein. The other three lanes contained unnormalized samples from transfected cells, as well as a mock transfection as a control. The dark bands within each lane demonstrate the presence of proteins.markerMAP1B-AMAP1B-BMAP1B-HMockSamplesolve for xug/ul prot20ugto 90ul w BCLB1000ugto 1000ul with BCLB+EGFP0.43.30.360.030.02998.5-1998.5MAP1B0.99.61.020.769.31036.6-36.6MAP1B+EGFP0.21.90.2107.5-17.55377.1-4377.1

Figure 4: Protein Assay. In order to normalize whole cell extract samples a protein assay must conduct to identify the amount of protein in each sample. A) Depicts a graph of a normal curve from a protein assay. This was conducted by preparing known sample with specific concentrations of protein and running these samples through a Biophotometer. To prepare these samples BCLB+ and BSA were combined in specific concentration, then Bradford Reagent was added. Bradford reagent will dye the proteins blue, this blue dye is what the Biophotometer detects to determine the protein concentration. B) This data is used to normalize the samples before they are run through a Western Blot. It utilizes the equation determined in Figure 4A to solve for the concentration of the unknown samples and how much BCLB+ needs to be added in order for them to all have the same protein concentration for 90ul samples. For this protein assay EGFP required 60ul of sample and 30 ul of BCLC+, MAP1B required 21ul sample and 69ul of BCLB+, and MAP1B+EGFP required 90ul of sample and 0ul of BCLB+. Not normalized samples were also prepared in which each sample had 90ul of sample and no BCLB+. A)B)Figure 5: One minute exposure of Nitrocellulose Membrane. To identify if MAP1B was successfully expressed a Western Blot was run with the samples: MAP1B+EGFP, MAP1B, EGFP, MAP1B+EGFP not normalized, MAP1B not normalized, and EGFP not normalized. The first lane was filled with marker/ladder protein and the subsequent lanes were filled respectively. The SDS-PAGE gel from the Western blot was transferred onto a Nitrocellulose membrane, blocked with milk in TBST, and incubated with the primary antibody GFP and secondary antibody anti-rabbit GFP. Upon being exposed for 1 minute we were able to successfully express MAP1B in the normalized and not normalized samples. This is indicated by the blue arrows.

Marker

B)MAP1Bmicrotubule associated proteinbelieved to play a role in cytoskeletal changes of neurite extensionsmolecular function: protein and microtubule bindingplasmid is tagged with GFP on the N terminal of the backbone. (GFP is a fluorescence tag)bacterial resistance to kanamycinmolecular weight: 270,634 Da

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