Proteomic identification of ZO-1/2 as a novel scaffold for Src/Csk regulatory circuit.
To elucidate the regulatory mechanism of cell transformation induced by c-Src tyrosine kinase, we performed a proteomic analysis of tyrosine phosphorylated proteins that interact with c-Src and/or its negative regulator Csk. The c-Src interacting proteins were affinity-purified from Src transformed cells using the Src SH2 domain as a ligand. LC-MS/MS analysis of the purified proteins identified general Src substrates, such as focal adhesion kinase and paxillin, and ZO-1/2 as a transformation-dependent Src target. The Csk binding proteins were analyzed by a tandem affinity purification method. In addition to the previously identified Csk binding proteins, including Cbp/PAG, paxillin, and caveolin-1, we found that ZO-1/2 could also serve as a major Csk binding protein. ZO-2 was phosphorylated concurrently with Src transformation and specifically bound to Csk in a Csk SH2 dependent manner. These results suggest novel roles for ZO proteins as Src/Csk scaffolds potentially involved in the regulation of Src transformation.
Saito
Kazunobu
K
Department of Oncogene Research, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan.
Enya
Kengo
K
Oneyama
Chitose
C
Hikita
Tomoya
T
Okada
Masato
M
D000595:Amino Acid Sequence; D000818:Animals; D000081247:CSK Tyrosine-Protein Kinase; D002460:Cell Line; D002645:Chickens; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D051381:Rats; D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein; D018909:src Homology Domains; D019061:src-Family Kinases
D008565:Membrane Proteins; D010750:Phosphoproteins; C569042:TJP1 protein, human; C569046:TJP2 protein, human; C569043:Tjp1 protein, mouse; C569044:Tjp1 protein, rat; C569047:Tjp2 protein, mouse; D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human