Identification of TARP Phosphorylation Sites and Their Significance in the Brain
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1 Identification of TARP Phosphorylation Sites and Their Significance in the Brain Susumu SHM BE17 Department of Cellular and Molecular Physiology NIDA Neuroproteomics Center, December 13, 2008
2 Glutamate NMDA receptor Mg 2+ AMPA receptor TARPs Ca 2+, Na + PSD Na + Transmembrane AMPAR Regulatory Proteins ( -2/stargazin, -3, -4, -8 and -5, -7)
3 Loss of AMPAR in hippocampus of TARP/ -8 KOs GluR1 staining in Wt. GluR1staining in 8 KO Rouach N. et al. (2005) Nat. Neurosci.
4 TARP/Stargazin is phosphorylated in brain Stargazin Stargazin PSD95 Tubulin Synapto physin PPase Synapto physin PPase - + Tomita et al (2005) Neuron
5 CaMKII and PKC are involved in stargazin phosphorylation in neurons Stargazin Stargazin Tomita et al (2005) Neuron
6 AMPA receptor TARPs NMDA receptor PSD95 PSD Ca 2+ CaMKII PKC X X
7 Amino acid sequence of stargazin cytoplasmic domain DRHKQLRATA RATDYLQASA ITRIPSYRYR YQRRSRSSSR STEPSHSRDA SPVGVKGFNT LPSTEISMYT LSRDPLKAAT TPTATYNSDR DNSFLQVHNC IQKDSKDSLH ADTANRRTTPV Phosphorylate recombinant proteins in vitro Digest by trypsin Fraction by HPLC MASS-spec
8 Both CaMKII and PKC directly phosphorylate stargazin cytoplasmic domain in vitro CaMKII PKC Coomassie Brilliant Blue
9 HPLC P Da, corresponding to residues with one phosphorylation site (IPpSYR)
10 Radio Edman sequencing
11 DRHKQLRATA RATDYLQASA ITRIPSYRYR YQRRSRSSSR STEPSHSRDA SPVGVKGFNT LPSTEISMYT LSRDPLKAAT TPTATYNSDR DNSFLQVHNC IQKDSKDSLH ADTANRRTTPV
12 DRHKQLRATA RATDYLQASA ITRIPSYRYR YQRRSRSSSR STEPSHSRDA SPVGVKGFNT LPSTEISMYT LSRDPLKAAT TPTATYNSDR DNSFLQVHNC 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 R Y Q R R S2R S3S4S5R S6T E P S7H S8R D A S9P V G V K G F N T L 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 R D 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
13 Alternative approach with 2D gel DRHKQLRATA RATDYLQASA ITRIPSYRYR YQRRSRSSSR STEPSHSRDA SPVGVKGFNT LPSTEISMYT LSRDPLKAAT TPTATYNSDR DNSFLQVHNC IQKDSKDSLH ADTANRRTTPV Purified recombinant STG-mut In vitro phosphorylation by CaMKII and PKC TPCK-Trypsin: K-X; R-X 2D gel (1st Electrophoresis, 2nd PLC)
14 PKC phosphorylation sites wild type S9A (s )( ) -IPS1YR- -S2R- -S3S4S5R Chrom matography PKC Electrophoresis S3S4S5R- -S6TEPS7HS8R- -S3S4S5R- -S6TEPS7HS8R- -DAS9PVGVK PKC
15 CaMKII phosphorylation sites CaMKII Chromatography Electrophoresis - -DAS9PVGVK- -S3S4S5R- -S3S4S5R- -S6TEPS7HS8R- -S6TEPS7HS8R wild type S9A (s ) -IPS1YR- -S2R- -S3S4S5R- CaMKII
16 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 R Y Q R R S2R S3S4S5R S6T E P S7H S8R D A S9P V G V K G F N T L 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 R D 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 plasticity Genetic mouse model Electrophysiological analysis Isolation of P-specific binding proteins AMPA receptor TARPs NMDA receptor PSD95 PSD Ca 2+ CaMKII PKC X X
17 Acknowledgements Tomita Lab. Kwang Kim NIDA Neuroproteomics Center / KECK facility Kathryn Stone Mary Lpresti J. Myron Crawford
18
19 Stargazin phosphorylation in cerebro-cortical neurons Phospho amino acid analysis The image cannot be displayed. Your computer may not have enough memory to open the image, or the image may have been corrupted. Restart your computer, and then open the file again. If the red x still appears, you may have to delete the image and then insert it again. Stargazin Cerebro-cortical neuron Stargazin transfected CHO cells
20 Nine serine residues in stargazin are phosphorylated. Wild type -IPS 1 YR- -S 2 R- -S 3 S 4 S 5 R- -S 6 TEPS 7 HS 8 R- -S 6 TEPS 7 HS 8 R- -DAS 9 PVGVK- S9A(S )
21
22 Phospho-mimic stargazin (S9D) enhances the AMPA EPSCs DIC GFP-fluorescence Overlay Tomita et al (2005)
23 Phosphorylation of stargazin is necessary for LTP Control S9A
24
25 Generate P-specific antibodies E S EQ U ENCE PRO JECT N AME PR O JECT# TRI P( P-S)YRYR - C PHOSPHO STARGAZIN ( S22 8) PKC SITE AB TRI PSYRYR - C STARGAZIN ( S22 8) PKC SIT E CONT ROL AB RRSR( P-S)S(P-S)RSTE - C PHOSPHO STARGAZIN (S239/241)CAMKII AB RRSRSS( P-S)R STE - C PHOSPHO STARGAZIN (S241 ) CAMKII AB RRSR( P-S) SSRSTE - C PHOSPHO STARGAZIN ( S239) CAMKII AB RRSRS( P-S)SRSTE - C PHOSPHO STARGAZIN (S240)PKC/CAMKII AB RRSRSSSRSTE - C STARGAZIN (S239/240/241 ) CAMKII-CONTROL AB15436 YQR RSRSSSRSTE SSRSTE - C STARGAZIN (S237/239/240/241 ) PKC/CAMKII - 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 AB YQR R( PS)RS( PS)SR STE - C STARGAZIN (S237/240) PKC/CAMKII AB YQR R( PS)RSS( PS)R STE - C STARGAZIN (S237/241) PKC/CAMKII AB YQR RSR( PS)(PS)SR STE - C STARGAZIN (S239/240) PKC/CAMKII AB YQR RSRS( PS) ( PS)R STE - C STARGAZIN (S240/241) PKC/CAMKII AB YQR R( PS)RSSSRSTE - C STARGAZIN (S237) PKC/CAMKII AB
26 Dot blotting with P- & non-p peptides Kwang Kim
27 Dot blotting with P- & non-p peptides Kwang Kim
28
29 9 sites x (A or D) = 2 9 = 512 Categorize each sites by kinases. 2 classes (total 9 sites) x (A or D) = 4! NMDA induces dephosphorylation of stargazin Stargazin min Tomita et al (2005)
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