Physical and functional interactions between Lyn and p34cdc2 kinases in irradiated human B-cell precursors.
Exposure of human B-cell precursors (BCP) to ionizing radiation results in cell cycle arrest at the G2-M checkpoint as a result of inhibitory tyrosine phosphorylation of p34cdc2 . Here, we show that ionizing radiation promotes physical interactions between p34cdc2 and the Src family protein-tyrosine kinase Lyn in the cytoplasm of human BCP leading to tyrosine phosphorylation of p34cdc2. Lyn kinase immunoprecipitated from lysates of irradiated BCP as well as a full-length glutathione S-transferase (GST)-Lyn fusion protein-phosphorylated recombinant human p34cdc2 on tyrosine 15. Furthermore, Lyn kinase physically associated with and tyrosine-phosphorylated p34cdc2 kinase in vivo when co-expressed in COS-7 cells. Binding experiments with truncated GST-Lyn fusion proteins suggested a functional role for the SH3 rather than the SH2 domain of Lyn in Lyn-p34cdc2 interactions in BCP. The first 27 residues of the unique amino-terminal domain of Lyn were also essential for the ability of GST-Lyn fusion proteins to bind to p34cdc2 from BCP lysates. Ionizing radiation failed to cause tyrosine phosphorylation of p34cdc2 or G2 arrest in Lyn kinase-deficient BCP, supporting an important role of Lyn kinase in radiation-induced G2 phase-specific cell cycle arrest. Our findings implicate Lyn as an important cytoplasmic suppressor of p34cdc2 function.
Uckun
F M
FM
Department of Therapeutic Radiology-Radiation Oncology, University of Minnesota Health Sciences Center, Minneapolis 55455, USA.
Tuel-Ahlgren
L
L
Waddick
K G
KG
Jun
X
X
Jin
J
J
Myers
D E
DE
Rowley
R B
RB
Burkhardt
A L
AL
Bolen
J B
JB
D000595:Amino Acid Sequence; D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology / Q000201:enzymology* / Q000528:radiation effects; D001665:Binding Sites / Q000235:genetics; D016203:CDC2 Protein Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D016195:G2 Phase / Q000528:radiation effects; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000201:enzymology / Q000528:radiation effects; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010766:Phosphorylation; D015452:Precursor B-Cell Lymphoblastic Leukemia-Lymphoma / Q000201:enzymology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry / Q000528:radiation effects; D019061:src-Family Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D019061:src-Family Kinases; D016203:CDC2 Protein Kinase