Visual Snapshots of Intramolecular PKC Activity at the Onset of Mitosis

MARM 200805/18/2008

Zhaohua DaiDepartment of Chemistry & Physical Sciences, NY

zdai@pace.edu

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 II in G2/M Transition

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

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

Target: lamin B Ser405

85K

Km (M): 4.9 (soluble) and 3.9 (envelope). IC50: 16 Mnocodazole

Chelerythrine

Chelerythrine (PKC inhibitor ????)

NBD-based Fluorescent Sensor for PKC

Phe Arg Arg Arg Arg Lys amide

NH

O

HO

N

N

O2N

ONBD-peptide

Yeh, R.-H.; Yan, X.; Cammer, M.; Bresnick, A. R.; Lawrence, D. S. J. Biol. Chem. 2002, 277, 11527-11532

Assay PKC PKC PKC

Radioact. 9.0±1.0 9.2 ±0.4 5.0 ±1.0

Fluoresc. 29 ±3 27 ±4 30 ±5

Km(M)

VIP

Caged PKC Sensor

Phe Arg Arg Arg Arg Lys amide

NH

O

O

N

N

O2N

O

O2N OCH3

OCH3

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

KVIP

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

Studying Mitosis

Microinjection

Phe Arg Arg Arg Arg Lys amide

NH

O

O

N

N

O2N

O

O2N OCH3

OCH3

PtK2 Cells: flat

Kangroo rat didneyepithelial cells

KVIP

VIP PKC Activity

Other mitotic kinases: Akt-1, AurB, Cdc-2, Plk1 (do not work on VIP) Nek2 (weakly)

S. Kumar

PKC in PtK2

before 0 min injection 2 min uncaging 3 min

Green Fl NBD

Red Fl70K dextran-Texas red

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

4 min 5 min 6 min 7 min

0 min injection 2 min uncaging 25 min

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

Mmc1.mov Mmc2.mov

Injection with 200 M KVIP before NEBD

1

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

2

-13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 8 9

t (min) relative to NEB

Re

lati

ve

Flu

ore

sc

en

ce

Total cellsNEBD Large

enhancement (>40%)

Small enhancement(<40%)

No enhancement

18 Yes 15 6 9 0

No 3 3

1.PKC activityaccompaniesNEBD.Which one?

2. PKC activitylevels off afterNEBD:

PKC off? orSensor gone?

0

0.2

0.4

0.6

0.8

1

1.2

0 2 4 6 8 10 12 14

Time (after NEBD)

I f

Injection with 200 M KVIP (Uncaging after NEBD)

Total cells Large enhancement (>40%)

Small enhancement(<40%)

No enhancement(within 5%)

Very smallEnhancement(within 15%)

16 0 0 14 2

1. No PKC activity right after NEBD?2. Both PKC and phosphatase are active?

Right after uncaging

Texas red

Incubation with 1.5 M okadaic acid

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 2 4 6 8 10 12 14 16 18

No PKC activityright after NEBD.

Total cells NEBD Large enhancement (>40%) Small enhancement(<40%)

No enhancement Little enhancement(around 15%)

10 Yes 10 0

0 8 2

Phosphatase inhibited

High PKC inhibitor concentration (12 M) induced or blocked cells at prophase

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

IINek2

IC50 1.3 M 11 nM no obs. inhibition

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

S. Kumar

PKC , might be implicated in NEBD. Which one?

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

PKCIC50 (M) Ki (M)

0.0019 0.00080

PKC 385-fold PKC 580-fold

PKC 2730-fol PKC 600-fol

PKC 1310-fold PKC 1210-fold

PKC 940-fold PKC 640-fold

Arg Arg Gly Ala Leu Arg Dap Ala NHCH2CH2SH

NH C

O

N

Cl

Cl

HN

O

Ala

6

Lee, Nandy, Lawrence. JACS, 2004

Coinjection of 2 mM PKC inhibitor and 200 M KVIP

0

0.2

0.4

0.6

0.8

1

1.2

0 5 10 15 20 25 30 35

t(injection)

If

Total cells NEBD Large enhancement (>30%)

Small enhancement(<30%)

No enhancement

10 Yes 0 0 0 0

No 10 0 0 10

When PKCs areshutdown, NEBD is blocked w/o FLenhancement.

Texas-red30 min after inj

Co-injection of 1 M PKC inhibitor and 200 M KVIP

11.11.21.31.41.51.61.71.81.9

2

-17

-16

-15

-14

-13

-12

-11

-10

-9 -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5t (NEBD)

If

Total cells NEBD Large enhancement (>30%)

Small enhancement(<30%)

No ehancement(within 1%)

15 Yes 12 6 5 1

No 3 0 0 3

PKC is responsiblefor NEBD and FL

1 or 2?

PKC shutdown

14 min after inj

Texas-red

Redistribution of PKCI and PKCII

In Cell Cycle

N. G. Dulyaninova

1: associated w/ nucleusin interphase and prophase.

2: everywhere in interphase Partial relocation to nuclear boundary in prophase.Significant for NEBD?

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.

• PKC is implicated in NEBD of PtK2 cells.

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

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

Acknowledgement• Prof. David S. Lawrence (Einstein, UNC) Dr. Williem Veldhuyzen, Dr. Sandip Nandy Prof. Sanjai Kumar (Einstein, Queens College)• Prof. Anne R. Bresnick (Einstein) Dr. Natalya G. Dulyaninova Dr. Zhonghua (Alice) Li

NIH (DSL, ARB)Pace University:Startup FundScholarly Research Fund