Table 1.
List of proteins identified following tyrosine-phosphoproteome analysis of CRP-stimulated human platelets. Proteins from basal and CRP-stimulated platelets (10 μg/ml, 90 sec) were immunoprecipitated using an antibody to phosphotyrosine. Immunoprecipitated proteins were separated by 4-12% NuPAGE Bis-Tris gels and analyzed by MS. Band numbers correspond to Figure 1.
| Protein Name | ANa) | Band No. | Functionb) |
|---|---|---|---|
| Proteins known to be tyrosine phosphorylated by GPVI | |||
| α-actinin 1 | P12814 | 4 | Actin-binding protein. Tyrosine-phosphorylated in activated platelets. |
| ADAP [Adhesion- and degranulation-promoting adapter protein] (SLAP-130, FYB) | O15117 | 3 | No recognized function in GPVI signaling. Binds to SKAP-Hom and SLP-76. |
| Btk [Bruton’s tyrosine kinase] | Q06187 | 7 | Critical role in activation of PLCγ2 and possibly Ca2+ entry by GPVI. |
| c-Cbl [E3 ubiquitin protein ligase] | P22681 | 4 | Feedback inhibition of GPVI signaling. |
| Cortactin | Q14247 | 7 | Role in GPVI signaling is unknown; shown to regulate actin polymerization in other cells. |
| Dok-2 [Downstream of tyrosine kinases] | O60496 | 13 | Function in the GPVI cascade is not known. |
| Fes | P07332 | 5 | GPVI signaling is maintained in Fes-knockout mice. |
| Fyn | P06241 | 11 | Src family kinase: stimulates FcR γ-chain ITAM phosphorylation. |
| Hck [Hemopoietic cell kinase] | P08631 | 11 | Src family kinases regulate multiple steps in GPVI cascade. |
| Lyn | P07948 | 12 | Src family kinase which mediates phosphorylation of FcR -chain ITAM and feedback regulation of GPVI. |
| p38α MAP kinase | Q16539 | 14 | Serine-threonine kinase. Implicated in regulation of cPLA2. |
| PLCγ2 [Phospholipase C γ2] | P16885 | 2 | Generates second messengers 1,2-diacylglycerol and IP3. |
| SHIP [SH2 containing inositol-5-phosphatase] | O00145 | 2 | Hydrolyses PI 3-kinase product, PI 3,4,5P3 to PI 3,4P2. Feedback inhibition of GPVI signaling. |
| SKAP-HOM [Src kinase-associated phosphoprotein 55-related protein] | O75563 | 13 | GPVI signaling is maintained in SKAP-HOM knockout mice. |
| SLP-76 [SH2 domain-containing leucocyte protein of 76 kDa] | Q13094 | 9 | Critical role in activation of PLCγ2 by GPVI. Constitutively bound to Gads. |
| Src | P12931 | 10 | Src family kinases regulate multiple steps in GPVI cascade |
| Syk [Spleen tyrosine kinase] | P43405 | 8 | Critical role in GPVI signaling: binds to phosphorylated ITAM in GPVI-FcRγ-chain complex. |
| Vav 1 | P15498 | 4 | Play a critical role in GPVI mediated activation of PLCγ2. |
| Proteins not previously known to be tyrosine phosphorylated by GPVI | |||
| 14-3-3 protein ε | P42655 | 17 | Member of 14-3-3 family. |
| 14-3-3 protein ζ/δ | P29312 | 17 | Member of 14-3-3 family. |
| Annexin A2 | P07355 | 15 | Calcium/phospholipid-binding protein. |
| Annexin A5 | P08758 | 15 | Calcium/phospholipid-binding protein. |
| DIP-1 [Dia interacting protein-1] (SPIN90) | Q9NZQ3 | 6 | Adapter protein implicated in regulation of actin polymerization. |
| Gads [Grb2-related adaptor protein 2] | O75791 | 14 | Supports activation of PLCγ2 by GPVI. Constitutively bound to SLP-76. Tyrosine phosphorylation shown in this study. |
| G6f | Q7Z5H2 | 16 | Immunoglobulin membrane protein of unknown function. Binds Grb2. Phosphorylated on Y281 by GPVI (present study). |
| Myosin-9 | P35579 | 1 | Regulation of cell contractility. |
| PSTPIP2 [Proline-serine-threonine phosphatase-interacting protein 2] | Q9H939 | 14 | Involved in the regulation of actin polymerisation |
| RSP-1 [Ras suppressor protein 1] | Q15404 | 17 | Inhibits Ras signaling |
| Tight junction protein ZO-2 | Q9UDY2 | 2 | Plays a role in tight junctions. Contains one SH3 domain. |
| β-Pix [Rho guanine nucleotide exchange factor 7] | Q14155 | 7 | Interacts with PAK kinases through the SH3 domain. Interacts with c-Cbl. |
a)
AN indicates SWISS-PROT accession number.
b)
function either known or predicted by homology via SwissProt and NCBI.