Characterization of phosphotyrosine binding motifs in the cytoplasmic domain of platelet endothelial cell adhesion molecule-1 (PECAM-1) that are required for the cellular association and activation of the protein-tyrosine phosphatase, SHP-2

被引:127
作者
Jackson, DE
Kupcho, KR
Newman, PJ
机构
[1] BLOOD CTR SE WISCONSIN INC, BLOOD RES INST, MILWAUKEE, WI 53233 USA
[2] MED COLL WISCONSIN, DEPT PHARMACOL, MILWAUKEE, WI 53226 USA
[3] MED COLL WISCONSIN, DEPT CELLULAR BIOL, MILWAUKEE, WI 53226 USA
关键词
D O I
10.1074/jbc.272.40.24868
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Recent studies have shown that the Src homology-2 (SH2) domain containing protein-tyrosine phosphatase, SHP-2, associates with the cytoplasmic domain of PECAM-1 as it becomes tyrosine-phosphorylated during platelet aggregation: a process that can be mimicked in part by small synthetic phosphopeptides corresponding to the cytoplasmic domain of PECAM-1 encompassing tyrosine residues Tyr-663 or Tyr-686. To further examine the molecular requirements for PECAM-1/SHP-2 interactions, we generated human embryonic kidney (HEK)-293 cell lines that stably expressed mutant forms of PECAM-1 harboring tyrosine to phenylalanine (Tyr --> Phe) mutations in the cytoplasmic domain, Y663F and Y686F forms of PECAM-1 were tyrosine-phosphorylated to a somewhat lesser extent than wild-type PECAM-1, and a doubly substituted Y663,686F form of PECAM-1 failed to become tyrosine-phosphorylated, suggesting that the PECAM-1 cytoplasmic domain tyrosine residues 596, 636 and 701 do nob serve as substrates for cellular kinases, Interestingly, SHP-2 binding was lost when either Tyr-663 or Tyr-686 were changed to phenylalanine, indicating that both residues are required for SHP-2/PECAM-1 association, Although PECAM-1 phosphopeptides NSDVQpY(663)TEVQV and DTETVpY(686)SEVRK stimulated the catalytic activity of the phosphatase to a similar extent, surface plasmon resonance studies revealed that the Tyr-663 containing peptide had approximately 10-fold higher affinity for SHP-2 than did the Tyr-686 peptide. Finally, peptido-precipitation analysis showed that the NH2-terminal SH2 domain of SHP-2 reacted preferentially with the Tyr-663 PECAM-1 phosphopeptide, while the Tyr-686 phosphopeptide associated only with the COOH-terminal SH2 domain of the phosphatase, Together, these data provide a molecular model for PECAM-1/SHP-2 interactions that may shed light on the downstream events that follow PECAM-1-mediated interactions of vascular cells.
引用
收藏
页码:24868 / 24875
页数:8
相关论文
共 62 条
[1]   A WIDELY EXPRESSED HUMAN PROTEIN-TYROSINE PHOSPHATASE CONTAINING SRC HOMOLOGY-2 DOMAINS [J].
AHMAD, S ;
BANVILLE, D ;
ZHAO, ZZ ;
FISCHER, EH ;
SHEN, SH .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1993, 90 (06) :2197-2201
[2]   ENDOCAM - A NOVEL ENDOTHELIAL-CELL CELL-ADHESION MOLECULE [J].
ALBELDA, SM ;
OLIVER, PD ;
ROMER, LH ;
BUCK, CA .
JOURNAL OF CELL BIOLOGY, 1990, 110 (04) :1227-1237
[3]   PROTEIN-TYROSINE-PHOSPHATASE SHPTP2 COUPLES PLATELET-DERIVED GROWTH-FACTOR RECEPTOR-BETA TO RAS [J].
BENNETT, AM ;
TANG, TL ;
SUGIMOTO, S ;
WALSH, CT ;
NEEL, BG .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1994, 91 (15) :7335-7339
[4]  
BERMAN ME, 1995, J IMMUNOL, V154, P299
[5]  
Berman ME, 1996, J IMMUNOL, V156, P1515
[6]   ONCOGENES AND SIGNAL TRANSDUCTION [J].
CANTLEY, LC ;
AUGER, KR ;
CARPENTER, C ;
DUCKWORTH, B ;
GRAZIANI, A ;
KAPELLER, R ;
SOLTOFF, S .
CELL, 1991, 64 (02) :281-302
[7]  
CASE RD, 1994, J BIOL CHEM, V269, P10467
[8]  
DECHERT U, 1994, J BIOL CHEM, V269, P5602
[9]   Spatial constraints on the recognition of phosphoproteins by the tandem SH2 domains of the phosphatase SH-PTP2 [J].
Eck, MJ ;
Pluskey, S ;
Trub, T ;
Harrison, SC ;
Shoelson, SE .
NATURE, 1996, 379 (6562) :277-280
[10]   SH2-CONTAINING PHOSPHOTYROSINE PHOSPHATASE AS A TARGET OF PROTEIN-TYROSINE KINASES [J].
FENG, GS ;
HUI, CC ;
PAWSON, T .
SCIENCE, 1993, 259 (5101) :1607-1611