Physical and Functional Interactions between Lyn and p34 Kinases in Irradiated Human B-cell Precursors

Abstract

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.

STAT3 is a substrate of SYK tyrosine kinase in B-lineage leukemia/lymphoma cells exposed to oxidative stress

“…Immunoprecipitations, immune complex kinase assays (KA), cell-free KA, phosphoamino acid analysis, and WBA were performed as previously reported (7,8,15,16,26,27,(56)(57)(58). Antibodies against phospho-STAT3 (anti-phospho-STAT3[pY705) were obtained from Sigma-Aldrich.…”

“…The protective effect of STAT3 against apoptosis has been explained partly by upregulation of antiapoptotic proteins such as Bcl-XL and survivin as well as inactivation of caspases (12)(13)(14). It has been well established that OS induces the activation of various protein tyrosine kinases (PTK) but the PTK responsible for OS-mediated activation of STAT3 has not yet been identified (7,9,(15)(16)(17). SYK is a cytoplasmic PTK with multiple important regulatory functions in B-lineage lymphoid cells (18).…”

Spleen tyrosine kinase (Syk) deficiency in childhood pro-B cell acute lymphoblastic leukemia

“…Samples were immunoprecipitated with antisera prepared against SYK, LYN or BTK, as described (Mahajan et al, 1999;Uckun et al, 1996). The antisera were diluted and immune complexes collected by incubation with 15 ml protein A Sepharose.…”

Activation of Src in human breast tumor cell lines: elevated levels of phosphotyrosine phosphatase activity that preferentially recognizes the Src carboxy terminal negative regulatory tyrosine 530

“…Src speci®c activity was elevated two-to ®vefold in these tumor cell lines, relative to normal control cells. In order to obtain a second quantitative measurement of Src kinase activity in these cell lines, Src kinase activity from Elevated PTPase and Src kinase in breast tumor cells C Egan et al these lines was also tested by its ability to phosphorylate two peptide substrates, the Cdc2(6-20) peptide, which has been previously shown to be an excellent substrate for Src family kinases (Litwin et al, 1991;Uckun et al, 1996;Yuan et al, 1995) and the`Src optimal peptide' (AEEEIYGE-FEAKKKK) which has been shown to be an optimal substrate for Src kinase (Songyang et al, 1995). The relative amounts of phosphate incorporation in the peptide assays were measured by scintillation counting (Table 1) and the activity of Src in each cell line was again compared to HFF cells to determine the fold increase in activity, and the increase in speci®c activity.…”

“…Quantitation of band intensity was done by densitometer scan of the autoradiogram of the gel after appropriate exposures. To assay Src kinase activity against the Cdc2(6-20) peptide (Uckun et al, 1996;Yuan et al, 1995) and the Src optimal peptide (AEEEIYGEFEAKKKK) (Songyang et al, 1995), immunoprecipitates containing Src protein were resuspended in 50 ml of Cdc2 Assay Bu er (50 mM Tris pH 7.0, 0.05 mM Sodium Orthovanadate, 3.0 mM pNPP, 50 mM MgCl 2 , 5 mM MnCl 2 , 0.1% NP40, 0.08 mM Cdc2(6-20) peptide, 0.1 mM ATP and 0.02 mCi/ul 32 P gamma-ATP). Reactions were allowed to continue at 308C for 20 min and then the Cdc2(6-20) peptide reactions were stopped by the addition of 25 ml of 50% acetic acid.…”