1. OBJECTIVES OF STUDY
1. Confirm the interaction between MAP1B and Fyn
2. Determine whether Fyn SH2 domain phosphorylation alters MAP1B binding
HYPOTHESIS
MAP1B, a microtubule associated protein, and Fyn interact via Fyn’s SH2
domain.
EXPERIMENTAL APPROACH
Functional Investigation of the Novel Binding Partners Src family kinase Fyn and
Microtubule Associated Protein MAP1B
Alexandra Frank1 and Karen Hinkle1
1Department of Biology and Physical Education, Norwich University, Northfield VT 05663
2Department of Biology, University of Vermont, Burlington, VT 05405
CONCLUSIONS AND SIGNIFICANCE
ACKNOWLEDGEMENTS
ABSTRACT
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, SFK’s 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 Fyn’s 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 Fyn’s 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.
A) B) C)
Fyn and MAP1B
Interaction Studies
Transfection of HEK293 cells with
MAP1B, EGFP, mock
Cell lysis to extract proteins
Protein Assay and
normalization of cell extracts
Figure 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-PAGE
DNA 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.
marker
MAP1B-A
MAP1B-B
MAP1B-H
Mock
y = 0.0827x + 0.0952
0.
0.45
0.9
1.35
1.8
2.25
0 5 10 15 20 25
Sample solve for x ug/ul prot 20ug
to 90ul w
BCLB 1000ug
to 1000ul
with BCLB+
EGFP 0.4 3.3 0.3 60.0 30.0 2998.5 -1998.5
MAP1B 0.9 9.6 1.0 20.7 69.3 1036.6 -36.6
MAP1B+EGF
P 0.2 1.9 0.2 107.5 -17.5 5377.1 -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)
MAP1B
microtubule associated protein
believed to play a role in cytoskeletal changes of neurite extensions
molecular function: protein and microtubule binding
plasmid is tagged with GFP on the N terminal of the backbone. (GFP is a fluorescence tag)
bacterial resistance to kanamycin
molecular weight: 270,634 Da
D)