Identification of TARP Phosphorylation Sites and Their Significance in the Brain

Susumu Tomita @ SHM BE17

Department of Cellular and Molecular Physiology

NIDA Neuroproteomics Center , December 13, 2008

NMDA

Glutamate

AMPAMg2+

TARPsNMDAreceptor

AMPAreceptor

g TARPs

PSDNa+Ca2+, Na+

Transmembrane AMPAR Regulatory Proteins

(-2/stargazin, -3, -4, -8 and -5, -7)

Loss of AMPAR in hippocampus of TARP/-8 KOs

GluR1 staining in Wt.

GluR1staining in8 KORouach N. et al. (2005) Nat. Neurosci.

TARP/Stargazin is phosphorylated in brain

StargazinPSD95

Stargazin

Synapto

TubulinSynaptophysin

PSD95

y pphysin

PPase

p y

+-PPase

Tomita et al (2005) Neuron

CaMKII and PKC are involved in stargazin phosphorylation in neurons

Stargazin

Stargazin

Tomita et al (2005) Neuron

TARPAMPA receptor

TARPs NMDA receptor

PSDPSD95

Ca2+

XX

PKCCaMKII

Amino acid sequence of stargazin cytoplasmic domain

DRHKQLRATA RATDYLQASA ITRIPSYRYR YQRRSRSSSR STEPSHSRDA SPVGVKGFNTQLPSTEISMYT LSRDPLKAAT TPTATYNSDR DNSFLQVHNC IQKDSKDSLH ADTANRRTTPVQ Q

Phosphorylate recombinant proteins in vitroPhosphorylate recombinant proteins in vitro

Digest by trypsin

Fraction by HPLC

MASS-spec

Both CaMKII and PKC directly phosphorylate stargazin cytoplasmic domain in vitro

CaMKII

PKC

CoomassieB illi tBrilliant

Blue

HPLC

P32

715.2638 Da, corresponding to residues 223-227 with one phosphorylation site (IPpSYR)

Radio Edman sequencing

http://www2.bioch.ox.ac.uk/immunoch/PCFWebSite/Sequencing.

DRHKQLRATA RATDYLQASA ITRIPSYRYRDRHKQLRATA RATDYLQASA ITRIPSYRYRYQRRSRSSSR STEPSHSRDA SPVGVKGFNT LPSTEISMYT LSRDPLKAAT TPTATYNSDRLPSTEISMYT LSRDPLKAAT TPTATYNSDRDNSFLQVHNC IQKDSKDSLH ADTANRRTTPV

DRHKQLRATA RATDYLQASA ITRIPSYRYRYQRRSRSSSR STEPSHSRDA SPVGVKGFNTYQRRSRSSSR STEPSHSRDA SPVGVKGFNT LPSTEISMYT LSRDPLKAAT TPTATYNSDRDNSFLQVHNC IQKDSKDSLH ADTANRRTTPVDNSFLQVHNC IQKDSKDSLH ADTANRRTTPV

D R H K Q L R A T A R A T D Y L Q A S A I T R I P S1Y R Y RY Q R R S2R S3S4S5R S6T E P S7H S8R D A S9P V G V K G F N TL P S T E I S M Y T L S R D P L K A A T T P T A T Y N S D RD N S F L Q V H N C I Q K D S K D S L H A D T A N R R T T P V

Alternative approach with 2D gel

DRHKQLRATA RATDYLQASA ITRIPSYRYRYQRRSRSSSR STEPSHSRDA SPVGVKGFNTYQRRSRSSSR STEPSHSRDA SPVGVKGFNT LPSTEISMYT LSRDPLKAAT TPTATYNSDRDNSFLQVHNC IQKDSKDSLH ADTANRRTTPVDNSFLQVHNC IQKDSKDSLH ADTANRRTTPV

Purified recombinant STG-mutIn vitro phosphorylation by CaMKII and PKCIn vitro phosphorylation by CaMKII and PKCTPCK-Trypsin: K-X; R-X2D gel (1st Electrophoresis 2nd PLC)2D gel (1st Electrophoresis, 2nd PLC)

PKC phosphorylation sites

wild type S9A (s123456789) -S2R- -S3S4S5R--IPS1YR-

PKC 5

6

3

5

6

3

5

6

5

6

5

6

mat

ogra

phy

yp ( )

-DAS9PVGVK--S6TEPS7HS8R--S6TEPS7HS8R--S3S4S5R- -S3S4S5R-

12

34 4

12

31

2 4

12

3

12 4

34

Electrophoresis+

Chr

om

PKC4

3

5

6

45

6 6

53

5

6

3

54

6

41

2 21

43

12

34

12

34 1

2

CaMKII phosphorylation sites

wild type S9A (s123456789) -S2R- -S3S4S5R--IPS1YR-

CaMKII

Chr

omat

ogra

phy

1 2 3 4 5 6 1 2 3 4 5 6

1 23

45 6

1 23 4 5 6

1 23 4 5 6

-DAS9PVGVK--S6TEPS7HS8R--S6TEPS7HS8R--S3S4S5R- -S3S4S5R-

Electrophoresis+ -2 2

CaMKII1 2

34

56

1 2 3 4 5 6

1 2 3 4 5 61 2 3 4 5 6

1 3 4 5 61 2 1 2 3 1 2 3

D R H K Q L R A T A R A T D Y L Q A S A I T R I P S1Y R Y RY Q R R S2R S3S4S5R S6T E P S7H S8R D A S9P V G V K G F N TL P S T E I S M Y T L S R D P L K A A T T P T A T Y N S D RD N S F L Q V H N C I Q K D S K D S L H A D T A N R R T T P V

Molecular Mechanisms for synaptic plasticityMolecular Mechanisms for synaptic plasticityGenetic mouse modelElectrophysiological analysisElectrophysiological analysisIsolation of P-specific binding proteins

AMPA receptor TARPs NMDA receptor

PSDPSD95

Ca2+

XXPKC

CaMKII

Acknowledgements

Tomita LabTomita Lab.Kwang Kim

NIDA Neuroproteomics Center / KECK facilityp y

Kathryn StoneMar LprestiMary LprestiJ. Myron Crawford

Stargazin phosphorylation in cerebro-cortical neurons

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Phospho amino acid analysis

Stargazin

Cerebro-cortical neuron Stargazin transfected CHO cells

Nine serine residues in stargazin are phosphorylated.Wild type -IPS1YR- -S2R- -S3S4S5R-

S TEPS HS R S TEPS HS R DAS PVGVK S9A(S )-S6TEPS7HS8R- -S6TEPS7HS8R- -DAS9PVGVK- S9A(S123456789)

Phospho-mimic stargazin (S9D) enhances the AMPA EPSCs

GFP-fluorescenceDIC Overlay

Tomita et al (2005)

Phosphorylation of stargazin is necessary for LTP

Control

S9A

Generate P-specific antibodiesE

SEQ U ENCE PROJECT N AME PRO JECT#SEQ U ENCE PROJECT N AME PRO JECT#TRI P(P-S)YRYR - C PHOSPHO STARGAZIN ( S22 8) PKC SITE AB1 543 5 TRI PSYRYR - C STARGAZIN ( S22 8) PKC SIT E CONT ROL AB1 543 5 RRSR( P-S) S(P-S)RSTE - C PHOSPHO STARGAZIN (S239/241)CAMKII AB15436-1 RRSRSS(P-S)R STE - C PHOSPHO STARGAZIN (S241 ) CAMKII AB15436-3( ) ( )RRSR( P-S) SSRSTE - C PHOSPHO STARGAZIN ( S239) CAMKII AB15436-2 RRSRS(P-S)SRSTE - C PHOSPHO STARGAZIN (S240)PKC/CAMKII AB15438-8 RRSRSSSRSTE - C STARGAZIN (S239/240/241 ) CAMKII-CONTROL AB15436

YQR RSRSSSRSTE - CSTARGAZIN (S237/239/240/241 ) PKC/CAMKII - UNMODIFIED CONTROL AB15438YQR RSRSSSRSTE - C UNMODIFIED CONTROL AB15438

RDA( P-S)PVGVK - C PHOSPHO STARGAZIN (S253)OTHER KINASE AB15437 RDA S PVGVK - C PHOSPOSTARGAZIN (S253) OTHER KINASE-CONTROL AB15437 YQR R( PS)R(PS)SSRSTE - C STARGAZIN (S237/239) PKC/CAMKII AB15438-2 YQR R( PS)RS(PS)SR STE - C STARGAZIN (S237/240) PKC/CAMKII AB15438-3 YQR R( PS)RSS(PS)R STE - C STARGAZIN (S237/241) PKC/CAMKII AB15438-4 YQR RSR(PS) (PS)SR STE - C STARGAZIN (S239/240) PKC/CAMKII AB15438-5 YQR RSRS( PS) (PS)R STE - C STARGAZIN (S240/241) PKC/CAMKII AB15438-6 YQR R( PS)RSSSRSTE - C STARGAZIN (S237) PKC/CAMKII AB15438-7

Dot blotting with P- & non-P peptides

Kwang Kim

Dot blotting with P- & non-P peptides

Kwang Kim

9 sites x (A or D) = 29 = 512 ……( )

C t i h it b kiCategorize each sites by kinases.

2 classes (total 9 sites) x (A or D) = 4 !( ) ( )

NMDA induces dephosphorylation of stargazinNMDA induces dephosphorylation of stargazin

min

Stargazin

min

Tomita et al (2005)