Sumit Saha's Studies of His-133 Phosphorylation on Profilin-1 and how it changes both the secondary structure and poly-L-proline binding site.

1. Possible Role of His-133
Phosphorylation on GActin Regulation
Sumit Saha

2. INTRODUCTION

3. Background
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Human profilin (PFN1) is an actin-binding
protein with involvement in
restructuring the actin cytoskeleton and
has shown to be a possible area of study
to decrease breast cancer metastasis
Profilin-1 is constructed around an
antiparallel beta-pleated sheet which is
between two alpha-helix segments
The His-133 residue is located in an area
of high solvent exposure and in between
alpha-helix 1 and 4
In the poly-L-proline binding site there is
not only the His-133, but also Tyr-6, Tyr139, Trp-3, and Trp-31
Figure 1: Ribbon Diagram of Profilin1

4. Background
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It is expected that given a
phosphorylation of the His-133,
there be dislocations of the other
four residues
His-133 is located on alpha-helix 4
near the C-terminus and is semiburied in a hydrophobic groove; this
groove functions as the polyproline
ligand binding site
Figure 1: Ribbon Diagram of Profilin1

5. Background
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Profilin has been proposed to
regulate several mechanisms of
actin.
The 1:1 complex that profilin
creates when bound to actin, shows
to change the conformation and
structure of the actin protein
sequestering actin from other
monomers
This allows increased actin
availability in the cytosol
Figure 2: Ribbon diagram of Profilin1 and Actin

6. Background
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Actin has shown to have a significant role in cancer metastasis
○ G-actin dynamically localizes to the leading edge of metastatic
cancer cells.
○ This localization augments cell movement due to an increase in Gactin concentrations
○ Actin has shown to play a significant position in cytokinesis during
the last phases of cell division
○ During cytokinesis, a contractile ring is formed by actin and myosin
allowing cleavage of the original single cell into two identical
offspring
○ It has been shown in the yeast Schizosaccharomyces pombe that in
this contractile ring is an active site for actin polymerization. This
polymerization can only occur, however, through the use of profilin1

7. Significance
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A large percentage of cancer patients are in stage IV in which the
cancer is metastatic due to the high density of blood and lymph
vessels in that area allowing the cancer to spread
Chemotherapy has shown to only minimally destroys cancer cells
(only 2.1% effective toward a five year survival)
Alternative approaches such as inhibition of uniquely modified
proteins such as human profilin with phosphorylated amino acids
could lead to a more efficient treatment option
A protein based approach, such as phosphorylated profilin protein
might result in a broad -based treatment of breast cancer
metastasis, since this approach would inhibit the tumor locomotion
and division.

8. Literature Review
➢ G-actin was seen to have a significant relationship with the
cell motility of cancer cells
➢ It is seen that G-actin dynamically localizes itself to the
leading edge of growing neuroblastomas and thus causes
great cell movement of the carcinoma [6]
➢ Profilin’s interaction with actin was also seen in the
contractile ring of actin and myosin since actin
polymerization during cytokinesis of cell division [11]
➢ Phosphorylated profilin has been shown under in vivo
conditions to have increased affinity to poly-L-proline
sequences in vitro and in vivo. [12]

9. Problem & Hypothesis
Research Problem: This project uniquely investigates the
conformational change of the profilin-1 protein by
phosphorylation of His-133. Conformational change of the
profilin-1 was applied to find possible changes in actinprofilin interactions.
Research Hypothesis: It was predicted that there be a
significant change in the secondary and tertiary structure
of the profilin-1 with addition of the phosphohistidine

10. MATERIALS & METHODS

11. Materials & Methods
Effect of Potassium Phosphoramidate on Secondary Structure of
Profilin: All CD spectra were collected on a Jasco 720
spectropolarimeter at room temperature in either 1 mm or 5mm path
length cuvettes. CD was done with four 1mL samples of increasing
concentrations of phosphoramidate from wavelengths of 190 to 230 nm.
Effect of Potassium Phosphoramidate on Tertiary Structure of
Profilin: Data were collected on the same four 1mL samples of
increasing concentration of potassium phosphoramidate using a Photon
Technology International (PTI) spectrofluorometer model QM-4/2005.
The slit-widths were set as 2 nm and the excitation wavelengths were set
at 290 nm and 280 nm in context to tryptophan and tyrosine, respectively.
The emission spectrum was recorded from 300 to 400 nm in 0.5
increments.

12. RESULTS

13. Figure 1: CD of Profilin-1 wild type
Figure 2: Gel Electrophoresis
of Profilin Protein

14. Figure 3: CD of PFN1 with
Increasing Concentration of
Potassium Phosphoramidate (1ul
PFN1/10uL Solution)
Figure 4: CD of PFN1 and
Phosphorylated His-133 PFN1

15. Figure 5: Fluorescence of PFN1
with Increasing Concentration of
Potassium Phosphoramidate at
290nm excitation wavelength
Figure 6: Fluorescence of PFN1
and Phosphorylated His-133 PFN1
at 290nm excitation wavelength

16. Figure 7: Fluorescence of PFN1
with Increasing Concentration of
Potassium Phosphoramidate at
280nm excitation wavelength
Figure 8: Fluorescence of PFN1
and Phosphorylated His-133
PFN1 at 280nm excitation
wavelength

17. DISCUSSION

18. Discovery
➢ This projects finds that with the addition of the
phosphohistidines, profilin-1 undergoes a change
in secondary structure and specifically in the polyL-proline binding site.
➢ These results show potential to regulating G-actin
as G-actin must bind to profilin-1 to perform
processes in locomotion and cytokinesis

19. Further Research & Limitations
➢ In response to this research, interactions between profilin-1
with the phosphohistidines and actin should be viewed to
see changes from normal interactions
➢ The effect of the phosphohistidines should be monitored in
vivo to see if the interaction indeed inhibits cancer growth
and movement.
➢ If inhibition of profilin-actin interactions were to occur, it
would harm both cancer and somatic cells. Further research
should focus on isolating the protein action to only cancer
cells.

20. Analytical Results
➢ The profilin migrates to the 15 kD band, the expected
molecular weight
➢ The CD curve displays characteristic relative minimas at
208nm 222nm, and characteristic absolute minimum at
215nm, confirming the secondary structure of profilin.
➢ There is a consistent trend of a growing minimum as the
concentration of the potassium phosphoramidate
increases. Since the minima of the profilin-1 depict the
alpha helices and beta pleaded sheets, it is conclusive
that the phosphoramidate influenced the secondary
structure of the protein

21. Analytical Results
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The greatest change in secondary structure was seen in the sample
with a 1:1 (100uL:100uL) volumetric ratio between profilin and
potassium phosphoramidate. The greatest percent change in
structure was 19.05%
This difference gives insight to a change in the secondary structure
of the protein due to the combination of effects from
phosphohistidines 133 and 119
Alpha-helix 4 residues (on which His-133 is located) interact
directly with beta-sheet of profilin by side chain-side chain
contacts [8]. Thus, there are possible structural perturbations of
the Arg 135 (alpha-helix 4) and Phe 83 (beta-strand 5)

22. Analytical Results
➢ The left maxima of the profilin do not show change reflecting
stability of the profilin with increasing concentrations of the
phosphoramidate
➢ The excitation wavelength of 290 nm and 280nm show change
in the tryptophan and tyrosine residues, respectively
➢ There is a constant trend in which the fluorescence signal
measures a greater maximum with addition of the
phosphohistidines
➢ Isolation of the greatest and most consistent maxima, as seen
in the sample with a 1:1 volumetric ratio between profilin and
phosphoramidate, shows a percent change of 14.6%

23. Analytical Results
➢ This change significantly indicates that the aromatic amino
acid tryptophan might have moved a slight distance away
from the phosphohistidine
➢ The poly-L-proline binding site remains hydrophobic and
intact as suggested by fluorescence maximum at 344 nm
➢ This residue movement may also shed light on more changes
in the poly-L-proline site since the chemical shift of each
residue is influenced by the local environment

24. References
[1] Das, T., Bae, Y. H., Wells, A., & Roy, P. (2008, October 20). Profilin-1 overexpression upregulates
PTEN and suppresses AKT activation in breast cancer cells. In Journal of Cellular Physiology.
Retrieved November 12, 2013, from Wiley Online Library.
[2] Goldschmidt-Clermont, P. J., Machesky, L. M., Doberstein, S. K., & Pollard, T. D. (1991, June 1).
Mechanism of the interaction of human platelet profilin with actin. In Rockefeller University Press.
Retrieved November 12, 2013
[3] Hayes, D. F. (n.d.). Patient information: Treatment of metastatic breast cancer (Beyond the Basics).
In UpToDate. Retrieved November 12, 2013
[4] Janke, J., Schluter, K., Jandrig, B., Theile, M., Kolble, K., Arnold, W., & Grinstein, E. (2000, May 8).
Suppression of Tumorigenicity in Breast Cancer Cells by the Microfilament Protein Profilin 1. In
Rockefeller University Press. Retrieved November 12, 2013
[5] Medzihradszky, K. F., Phillipps, N. J., Senderowicz, L., Wang, P., & Turck, C. W. (1996, November
13). Synthesis and characterization of histidine-phosphorylated peptides . In Protein Science. Retrieved
November 12, 2013, from Wiley Online Library.
[6] Lee, C. W., Vitriol, E. A., Shim, S., Wise, A. L., Velayutham, R. P., & Zheng, J. Q. (2013, June 17).
Dynamic Localization of G-Actin during Membrane Protrusion in Neuronal Motility. In Current Biology.
Retrieved November 12, 2013
[7] Martino, J. (2013, April 11). Chemotherapy Ineffective 97% of the Time. In Collective Evolution.
Retrieved November 12, 2013

25. References
[8] McLachlan, G. D., Cahill, S. M., Girvin, M. E., & Almo, S. C. (2006, May 14). Acid-Induced Equilibrium Folding
Intermediate of Human Platelet Profilin. In Albert Einstein College of Medicine. Retrieved November 12, 2013
[9] Mitchison, T. J., & Cramer, L. P. (1996, February 9). Actin-Based Cell Motility and Cell Locomotion. In Cell.
Retrieved November 12, 2013, from ScienceDirect.
[10] Pantaloni, D., & Carlier, M. (1993, December 3). How profilin promotes actin filament assembly in the
presence of thymosin β4. In Cell. Retrieved November 12, 2013
[11] Pelham, R. J., & Chang, F. (2002, June 27). Actin dynamics in the contractile ring during cytokinesis in fission
yeast. InNature. Retrieved November 12, 2013
[12] Sathish, K., Padma, B., Munugalavadla, V., Bhargavi, V., Wasia, R., Sairam, M., & Singh, S. S. (2004, May).
Phosphorylation of profilin regulates its interaction with actin and poly (l-proline). In Cellular Signalling. Retrieved
November 27, 2013, from Science Direct
[13] Schutt, C. E., Myslik, J. C., Rozycki, M. D., Goonesekere, N., & Lindberg, U. (n.d.). The structure of crystalline
profilin-beta-actin. In Princeton University. Retrieved November 12, 2013
[14] Shao J, Diamond MI (2012) Protein Phosphatase 1 Dephosphorylates Profilin-1 at Ser-137. PLoS ONE 7(3):
e32802. doi:10.1371/journal.pone.0032802
[15] Shao, J., Welch, W. J., DiProspero, N. A., & Diamond, M. I. (2008, June 23). Phosphorylation of Profilin by
ROCK1 Regulates Polyglutamine Aggregation. In American Society for Microbiology. Retrieved November 12,
2013
[16] What are the key statistics about breast cancer? (n.d.). In American Cancer Society. Retrieved November 12,
2013