Dok-R mediates attenuation of epidermal growth factor-dependent mitogen-activated protein kinase and Akt activation through processive recruitment of c-Src and Csk.
Dok-R has previously been shown to associate with the epidermal growth factor receptor (EGFR) and become tyrosine phosphorylated in response to EGF stimulation. The recruitment of Dok-R to the EGFR, which is mediated through its phosphotyrosine binding (PTB) domain, results in attenuation of mitogen-activated protein kinase (MAPK) activation. Dok-R's ability to attenuate EGF-driven MAPK activation is independent of its ability to recruit rasGAP, a known attenuator of MAPK activity, suggesting an alternate Dok-R-mediated pathway. Herein, we have determined the structural determinants within Dok-R that are required for its ability to attenuate EGF signaling and to associate with c-Src and with the Src family kinase (SFK)-inhibitory kinase, Csk. We demonstrate that Dok-R associates constitutively with c-Src through an SH3-dependent interaction and that this association is essential to Dok-R's ability to attenuate c-Src activity and diminish MAPK and Akt/PKB activity. We further illustrate that EGF-dependent phosphorylation of Dok-R requires SFK activity and, more specifically, that SFK-dependent phosphorylation of tyrosine 402 on Dok-R facilitates the inducible recruitment of Csk. We propose that recruitment of Csk to Dok-R serves to bring Csk to c-Src and down-regulate its activity, resulting in a concomitant attenuation of MAPK and Akt/PKB activity. Furthermore, we demonstrate that Dok-R can abrogate c-Src's ability to protect the breast cancer cell line SKBR3 from anoikis and that an association with c-Src and Csk is required for this activity. Collectively these results demonstrate that Dok-R acts as an EGFR-recruited scaffolding molecule that processively assembles c-Src and Csk to attenuate signaling from the EGFR.
Van Slyke
Paul
P
Sunnybrook and Women's Research Institute, 2075 Bayview Avenue, Research Building S-218, Toronto, Ontario, Canada M4N 3M5.
Coll
Mariano Loza
ML
Master
Zubin
Z
Kim
Harold
H
Filmus
Jorge
J
Dumont
Daniel J
DJ
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000818:Animals; D023102:Anoikis / Q000502:physiology; D001943:Breast Neoplasms / Q000378:metabolism; D000081247:CSK Tyrosine-Protein Kinase; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000037:antagonists & inhibitors* / Q000502:physiology; D066246:ErbB Receptors / Q000502:physiology; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D015398:Signal Transduction; D014443:Tyrosine / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; C115089:DOK2 protein, human; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D014443:Tyrosine; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D019950:Mitogen-Activated Protein Kinase 1