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Fluorescent Chemosensors for Biology: Visual Snapshots of Intramolecular Kinase Activity at the Onset of Mitosis Zhaohua Dai Department of Chemistry & Physical Sciences, NY. Research Interests. Fluorescent probes for kinase activity in live cells. Fluorescent and chiroptical

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Fluorescent Chemosensors for Biology: Visual Snapshots of Intramolecular Kinase Activity at the Onset of Mitosis Zhaohua DaiDepartment of Chemistry & Physical Sciences, NY


Research Interests

Fluorescent probes for kinase

activity in live cells

Fluorescent and chiroptical

probes for metal ions

Zn2+, Mn2+, Hg2+

Tyrosine Kinase, PKC

Das, D.; Dai, Z.; Holmes, A. E.; Canary, J. W. Chirality, 2008, 20, 585-591.

Dai, Z.; Canary, J. W. New J. Chem.2007, 31, 1708-1718.

Royzen, M.; Dai, Z.; Canary, J. W. J. Am. Chem. Soc.2005, 127, 1612-1613.

Dai, Z.; Xu, X.; Canary, J.W. Chirality2005, 17, S227-233.

Dai, Z.; Proni, G.; Mancheno, D.; Karimi, S.; Berova, N.; Canary, J.W. J. Am. Chem. Soc., 2004,126, 11760

Dai, Z.; Xu, X.; Canary, J. W. Chemical Communications2002, 1414-5.

Dai, Z.; Dulyaninova, N. G.; Kumar, S.; Bresnick, A. R.; Lawrence, D. S. Chem. & Biol.2007, 14, 1254-1260.

Wang, Q.; Dai, Z.; Cahill, S. M.; Blumenstein, M.; Lawrence, D. S. J. Am. Chem. Soc.2006, 128, 14016-14017.


Zinc in Brain

  • More Zn2+ in brain than in any other organ

  • Zn2+ and Cu2+ are implicated in Alzheimer’s, Parkinson’s, and Amyotrophic Lateral Sclerosis (ALS)

  • Complicated roles

  • Tools needed to image Zn2+ distribution and kinetics

High sensitivy

Poor Zn(II)/Cu(II) selectivity

TSQ, Zinquin


Tailoring Tripodal Ligands for Zinc Sensing

Zhaohua Dai and James W. Canary,  New J. Chem., 2007, 31, 1708-1718.


Chiral Fluorescent Probes for Zn2+

Higher Zn2+/Cu2+Selectivity

Stereochemical Control

2. Better contrast

Fertile Optical Information: Differential Circularly Polarized Fluorescence Excitation (CPE)


Stereochemical Approach to Improved Zn(II)/Cu(II) Selectivity

Zn2+ 11.0 7.1 8.95

Cu2+ 16.157.17.0

10-5 1 90*

logb

Zn2+/Cu2+

Selectivity:

15% acetonitrile/aqueous buffer pH 7.19

* Z. Dai, et al. unpublished


Fluorescence-detected Circular Dichroism (FDCD)

DF =

J-8100 Circular Dichroism System

with FDCD Attachment

Two channels of data

Nehira; Berova; Nakanishi; et al. J. Am. Chem. Soc. 1999, 121, 8681


Differential Circularly Polarized Fluorescence Excitation (CPE)

CPE utilized only DF part of FDCD raw data for analysis.

q: CD ellipticity; F: Fluorescence quantum yield.

Changes in DF will be very large when changes in BOTH fluorescence AND circular dichroism are large.


Zn2+

CPE Reduces Background from Free Ligand

Ellipticity  /mdeg

Relative Intensity If

Zn2+

/nm

/nm

[Zn(L)]2+

Zn2+

CPE F

Dai, Z.; Proni, G.; Mancheno, D.; Karimi, S.; Berova, N.; Canary, J.W. J. Am. Chem. Soc., 2004,126, 11760

Free ligand

/nm


CPE SELECTS AGAINST PROTEIN-BASED BACKGROUND FLUORESCENCE

Zn2+

Relative Intensity If

Ellipticity  /mdeg

Zn2+

Lysozyme

/nm

/nm

Lysozyme

CPEF

Lysozyme

+

[Zn(L)]2+

Zn2+

Dai, Z.; Proni, G.; Mancheno, D.; Karimi, S.; Berova, N.; Canary, J.W. J. Am. Chem. Soc., 2004,126, 11760

/nm


Chiral Fluorescent Sensor for Hg2+

We intend to use these ligands

to further develop CPE.


ColorimetricMn(II)Sensor

5-Br-PAPS-Zn(II)-EGTA

Displacement system


Achieved solid Zn(II)/Cu(II) selectivity through a stereochemical approach

Developed a new approach for analysis: CPE

CPE may be used to improve contrast in detecting metal ions by fluorescent, chiral ligands with low background

CPE may be used to diminish interference from fluorescent non-analytes

CPE needs further development

Summary for Metal Sensors


Caged Sensors for Kinase Activity

Snapshots of PKC Activity at the Onset of Mitosis

Light-Regulated Sampling of Protein Tyrosine Kinase Activity

Dai, Z.; Dulyaninova, N. G.; Kumar, S.; Bresnick, A. R.; Lawrence, D. S. Chem. & Biol.2007, 14, 1254-1260.

Wang, Q.; Dai, Z.; Cahill, S. M.; Blumenstein, M.; Lawrence, D. S. J. Am. Chem. Soc.2006, 128, 14016-14017.


Protein Kinase C

  • Cell proliferation, apoptosis, differentiation, migration

  • Cause cancer, etc.

  • Tools are needed for probing, therapeutics

Nakashima, S. J. Biochem.2002, 132, 669-675.


PKC in Early Mitosis (G2/M)

Review: Black, J. D. Front. Biosci.2000, 5, 406-423

P. Collas et al J. Cell Sci. 1999, 112, 977-987.


PKC bII in G2/M Transition

85K

Target: lamin B Ser405

nocodazole

Chelerythrine

Km (mM): 4.9 (soluble) and 3.9 (envelope).

IC50: 16 mM

Chelerythrine (PKC b inhibitor ????)

A. P. Fields et al. J. Biol. Chem.1994, 269, 19074-19080.

A. P. Fields et al. J. Biol. Chem.1996, 271, 15045-15053.


NBD-based Fluorescent Sensor for PKC

VIP

NBD-peptide

Km(mM)

Yeh, R.-H.; Yan, X.; Cammer, M.; Bresnick, A. R.; Lawrence, D. S.

J. Biol. Chem.2002, 277, 11527-11532


In vivo Studies in HeLa cells


Caged PKC Sensor

KVIP

Veldhuyzen, W. F. et alJ. Am. Chem. Soc.2003, 125, 13358-13359


Why Caged Sensors

  • In cuvette: investigator controls the start and stop of enzyme catalyzed rxns

  • In live cell: the cell controls the timing and during

  • Caged sensors can be delivered in inert forms and activated on demand

  • Give precise temporal control over sensor activity


Real-time temporal probing of PKC activity

Veldhuyzen, W. F. et alJ. Am. Chem. Soc.2003, 125, 13358-13359


Studying Mitosis

Microinjection

PtK2 Cells: flat

Kangroo rat didney

epithelial cells

KVIP


PKC in PtK2

S. Kumar


VIP PKC Activity

q*

Other kinases: Akt-1, AurB, Cdc-2, Plk1 (do not work on VIP)

Nek2 (weakly)

S. Kumar


Coinjection of 200 mM KVIP and 5 mM 70K dalton texas red-dextran

Green Fl

NBD

Red Fl

70K dextran-

Texas red

2 min uncaging

0 min injection

3 min

before


5 min

7 min

4 min

6 min


Coinjection of 200 mM KVIP and 5 mM 70K dalton texas red-dextran

0 min injection

2 min uncaging

25 min


Injection with 200 mM KVIP before NEBD

1.PKC activity

accompanies

NEBD.

Which one?

2. PKC activity

levels off after

NEBD:

PKC off?

or

Sensor gone?


0 min injection

2 min uncaging

11 min

Coinjection of 200 mM

KVIP and 5 mM 70K

dalton texas red-dextran

(uncaging after NEBD )


Injection with 200 mM KVIP (Uncaging after NEBD)

  • No PKC activity

  • right after NEBD?

  • 2. Both PKC and

  • phosphatase are active?


Incubation with 1.5 mM okadaic acid

Phosphatase inhibited

No PKC activity

right after NEBD.


High PKCb inhibitor concentration (12 mM) induced or blocked cells at prophase

a bII i m q z Nek2

1.3 mM 11 nM no obs. inhibition

IC50

S. Kumar

PKC a, b might be implicated

in NEBD. Which one?

65% of the cells (20 out of 31) are stuck at prophase

Tanaka, M. et al. Bioorg. Med. Chem. Lett. 2004, 14, 5171-5174


Coinjection w/ 2 mM PKCa inhibitor and 200 mM KVIP, 5 mM 70K Texas ted-dextran

PKCa

IC50 (mM) Ki (mM)

PKCb385-fold PKCg580-fold

PKCd 2730-fol PKCe 600-fol

PKCh 1310-fold PKCq 1210-fold

PKCi 940-fold PKCz 640-fold

0.0019 0.00080

Lee, Nandy, Lawrence. JACS, 2004


Coinjection w/ 2 mM PKCa inhibitor and 200 mM KVIP, 5 mM 70K rhodamine-dextran (No NEBD)

0 min injection

2 min uncaging

30 min


Coinjection of 2 mM PKCa inhibitor and 200 mM KVIP

When PKCs are

shutdown, NEBD

is blocked w/o FL

enhancement.


Co-injection of 1 mM PKCa inhibitor and 200 mM KVIP

2 min

5 min

9 min

3 min

6 min

13 min

0 min injection

4 min

7 min

14 min

Texas-red

fluorescence


Co-injection of 1 mM PKCa inhibitor and 200 mM KVIP

PKC a shutdown

PKC b is responsible

for NEBD and FL

b1 or b2?


Redistribution of PKCbI and PKCbIIIn Cell Cycle

b1: associated w/ nucleus

in interphase and prophase.

b2: everywhere in

interphase

Partial relocation to

nuclear boundary

in prophase.

Significant for NEBD?

N. G. Dulyaninova


Conclusion for Caged PKC Sensor

  • Caged sensors can be used to probe PKC activity at G2/M in live cells with temporal precision, providing a way to interrogate enzymatic activity at any point during the cell-division cycle.

  • PKCb is implicated in NEBD of PtK2 cells.

    It is active just prior to NEBD, not immediately after.


Acknowledgement

  • Prof. James W. Canary (NYU)

  • Prof. David S. Lawrence (Einstein, UNC)

  • Dr. Williem Veldhuyzen, Dr. Sandip Nandy

  • Prof. Sanjai Kumar

  • Prof. Anne R. Bresnick (Einstein)

  • Dr. Natalya G. Dulyaninova

  • Dr. Zhonghua (Alice) Li

  • Mike Isaacman

  • Cho Tan

  • Amanda Mickley

  • Patrick Carney

  • Nikhil Khosla

  • Pace Colleagues

  • Prof JaimeLee I. Rizzo

NSF (JWC) NIH (DSL, ARB, JWC)

Pace University (Startup Fund, Scholarly Research Fund, Kenan Award)


Mechanism of Uncaging


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