Interactions of p62(dok) with p210(bcr-abl) and Bcr-Abl-associated proteins.
A 62-kDa Ras GTPase-activating protein (RasGAP)-associated protein is tyrosine-phosphorylated under a variety of circumstances including growth factor stimulation and in cells transformed by activated tyrosine kinases. A cDNA for p62(dok), reported to be the RasGAP-associated 62-kDa protein, was recently cloned from Abl-transformed cells. In this study, the interactions of p62(dok) with Bcr-Abl and associated proteins were examined. In 32D myeloid cells and Rat-1 fibroblasts transformed by p210(bcr-abl), p62(dok) is tyrosine-phosphorylated and co-immunoprecipitates with Bcr-Abl, RasGAP, and CrkL, a Src homology 2 (SH2) and SH3 domain-containing adaptor protein. Tyrosine-phosphorylated p62(dok) from cells expressing p210(bcr-abl) bound directly to the SH2 domains of Abl and CrkL in a gel overlay assay. Previous work has shown that an SH2 domain deletion mutant of Bcr-Abl is defective in transforming fibroblasts but remains capable of inducing myeloid growth factor independence. In both fibroblasts and myeloid cells expressing this mutant, p62(dok) is underphosphorylated as compared with cells expressing full-length p210(bcr-abl) but remains capable of associating with Bcr-Abl. However, in a gel overlay assay, p62(dok) from cells expressing the SH2 domain deletion was incapable of associating directly with SH2 domains of Abl and CrkL. Interestingly, no direct binding between Bcr-Abl and p62(dok) could be demonstrated in a yeast two-hybrid assay. These data suggest that indirect interactions mediate the interaction between Bcr-Abl and p62(dok) and that the SH2 domain of Bcr-Abl is required for hyperphosphorylation of p62(dok). Further, hyperphosphorylation of p62(dok) correlates with the ability of Bcr-Abl to transform fibroblasts but not with the induction of growth factor independence in myeloid cells.
Bhat
A
A
Division of Hematology and Medical Oncology, Oregon Health Sciences University, Portland, Oregon 97201, USA.
Johnson
K J
KJ
Oda
T
T
Corbin
A S
AS
Druker
B J
BJ
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins; D016044:Fusion Proteins, bcr-abl / Q000378:metabolism*; D005982:Glutathione Transferase; D006801:Humans; D008954:Models, Biological; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D016601:RNA-Binding Proteins; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development; D017384:Sequence Deletion; D014162:Transfection; D018909:src Homology Domains
D004268:DNA-Binding Proteins; C489560:DOK1 protein, human; C074561:GAP-associated protein p62; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D016044:Fusion Proteins, bcr-abl