Inhibition of the Bcr-Abl oncoprotein by Bcr requires phosphoserine 354.
The BCR protein is involved in the inhibition of oncogenic activity of the Bcr-Abl oncoprotein. This inhibition is believed to be the result of binding to the SH2 domain of Bcr-Abl in a non-phosphotyrosine-dependent manner. We showed that the Arg to Leu mutation in the Phe-Leu-Val-Arg-Glu-Ser (FLVRES) sequence of the SH2 domain, known to interfere with phosphotyrosine sequence binding, did not block the binding of Bcr first exon sequences to the Abl SH2 domain. We examined the structural-functional properties of a first exon mutant of BCR lacking the oligomerization domain, termed Bcr(64-413), that encodes the Ser-Thr protein kinase activity of Bcr. The autokinase product contained a M(r) 45,000-47,000 and 55,000 protein. Both species were detected by a Bcr phosphoserine 354 sequence-specific antibody. In contrast, the S354A mutant of Bcr(64-413), although maintaining autokinase activity, produced only the M(r) 45,000-47,000 kinase product. Abl SH2 binding experiments indicated that the M(r) 55,000 species of Bcr(64-413) but not the M(r) 45,000-47,000 species bound strongly to glutathime transferase-Abl SH2. The S354A mutant of Bcr(64-413) did not bind to glutathime transferase-Abl SH2. An adenovirus encoding Bcr(64-413) S354A did not induce cell death in CML cell lines in contrast to wild-type Bcr(64-413). Our findings indicate that Ser-354 of Bcr is part of a gating mechanism, which, after its phosphorylation, allows structural changes to occur in the Bcr protein. This altered phosphoserine form of the Bcr protein selectively binds to the Abl SH2 domain of the oncoprotein, which we propose down-regulates the activity of the Bcr-Abl tyrosine kinase.
Hawk
Natalyn
N
Department of Molecular Pathology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
Sun
Tong
T
Xie
Shanhai
S
Wang
Yan
Y
Wu
Yun
Y
Liu
Jiaxin
J
Arlinghaus
Ralph B
RB
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D019556:COS Cells; D005091:Exons; D016044:Fusion Proteins, bcr-abl / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020014:K562 Cells; D008970:Molecular Weight; D009154:Mutation; D015513:Oncogene Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D010768:Phosphoserine / Q000276:immunology / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins; D051562:Proto-Oncogene Proteins c-bcr; D013329:Structure-Activity Relationship; D014162:Transfection; D018909:src Homology Domains / Q000235:genetics / Q000502:physiology
D000906:Antibodies; D015513:Oncogene Proteins; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D010768:Phosphoserine; D011505:Protein-Tyrosine Kinases; D016044:Fusion Proteins, bcr-abl; C496613:BCR protein, human; D051562:Proto-Oncogene Proteins c-bcr