Phosphotyrosine binding-mediated oligomerization of downstream of tyrosine kinase (Dok)-1 and Dok-2 is involved in CD2-induced Dok phosphorylation.
To date, five members of the downstream of tyrosine kinase (Dok) family have been characterized. In T cells, two members, Dok-1 and Dok-2, are expressed. CD2 or CD28 stimulation, but not CD3/TCR stimulation, induces Dok phosphorylation. Recent evidence suggests that they act as negative regulators of the CD2 and CD28 signaling pathways. To identify the molecular mechanisms involved in Dok-mediated inhibition, we have identified proteins that bind to the phosphotyrosine-binding (PTB) domain of Dok-1 and Dok-2. We showed that the Dok PTB domain mediates phosphotyrosine-dependent homotypic and heterotypic interactions of Dok-1 and Dok-2. Moreover, in CD2-stimulated Jurkat cells, Dok-1 coimmunoprecipitates with tyrosine-phosphorylated Dok-2. To study the involvement of PTB-mediated oligomerization in Dok function, we have generated Jurkat clones overexpressing Dok-1 or Dok-2 with a mutation that prevents oligomerization (in either the PTB domain or Tyr146 of Dok-1 and Tyr139 of Dok-2). These mutations abrogate CD2-induced phosphorylation and the ability of Dok-1 or Dok-2 to inhibit CD2-induced ERK1/2 and NFAT activation. Moreover, overexpression of Dok-1Y146F or Dok-2Y139F interferes with CD2-induced phosphorylation of endogenous Dok, whereas overexpression of PTB mutant or wild-type Dok does not. Taken together, these data indicate that PTB-mediated oligomerization of Dok-1 and Dok-2 represents an essential step for Dok phosphorylation and function.
Boulay
Iohann
I
Institut National de la Recherche Scientifique-Institut Armand-Frappier, Université du Québec, Laval, Canada.
Némorin
Jean-Guy
JG
Duplay
Pascale
P
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D019943:Amino Acid Substitution; D018801:CD2 Antigens / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D015536:Down-Regulation / Q000502:physiology; D006801:Humans; D019169:Jurkat Cells; D009154:Mutation; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism* / Q000502:physiology; D017434:Protein Structure, Tertiary; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D018801:CD2 Antigens; D004268:DNA-Binding Proteins; C489560:DOK1 protein, human; C115089:DOK2 protein, human; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D019000:Phosphotyrosine; D014443:Tyrosine