INTRODUCTION

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Mammalian cells respond to DNA damage with cell cycle arrest, activation of DNA repair, and, in the event of irreparable lesions, the induction of apoptosis. The signals controlling responses to genotoxic stress, while of importance to mutagenesis and treatment with certain anti-cancer agents, remain unclear. Certain insights, however, have been derived from the finding that DNA-damaging agents activate the c-Abl protein-tyrosine kinase (PTK) (1-4).¹ c-Abl is detectable in a nuclear complex with the DNA-dependent protein kinase (DNA-PK) and is activated in part in the response to ionizing radiation (IR) by a DNA-PK-dependent mechanism (5). Activation of c-Abl is associated with binding to the p53 tumor suppressor and the induction of growth arrest in G₁ phase through down-regulation of the cyclin-dependent kinase 2 (Cdk2) (6, 7). Other studies have demonstrated that c-Abl contributes to DNA damage-induced apoptosis (8, 9). These findings have supported a role for c-Abl in regulating the growth arrest and apoptotic responses to genotoxic stress.

c-Abl is present in a nuclear complex that includes the Src-like Lyn PTK (10). Lyn, like c-Abl, is activated by IR and other DNA damaging agents (11-14). The activation of nuclear Lyn by DNA damage is associated with binding of Lyn to Cdc2 (11-14). Lyn phosphorylates Cdc2 on Tyr-15 and thereby inhibits Cdc2 activity (11-14). Whereas activation of Cdc2 in a complex with cyclin B is required for the transition of cells from G₂ to M phase (15), inhibition of Cdc2 by Lyn could contribute in part to the arrest at G₂/M phase following exposure to DNA damaging agents. Alternatively, binding of Lyn to Cdc2 may prevent the interaction of Cdc2 with proteins such as c-Src that play a functional role in mitosis (16). Other studies have indicated that the activation of Lyn is not restricted to cells in G₂. In this context, arrest of cells in G₁/S phase by 1--D-arabinofuranosylcytosine is associated with activation of Lyn and binding of Lyn to Cdk2 (17). Thus, the available evidence suggests that the Lyn PTK, like c-Abl, plays a role in the cell cycle arrest response to DNA damaging agents.

DNA-PK, a complex of three proteins, is involved in the repair of DNA double-stranded breaks, V(D)J recombination, and transcription (18-22). The 470-kDa catalytic subunit of DNA-PK (DNA-PK_cs) is activated by binding with the 70- and 80-kDa Ku heterodimer to sites of DNA damage (23-26). Under some conditions, DNA-PK is a self-contained kinase that is activated by direct interaction with double-stranded DNA, whereas Ku stabilizes binding of DNA-PK to DNA ends (21, 27). Among the many substrates of activated DNA-PK_cs are p53, c-Abl, and Cdc2 (5, 19, 26). Autophosphorylation of DNA-PK_cs inhibits its activity by inducing the dissociation of DNA-PK_cs from DNA (28). Phosphorylation of DNA-PK_cs by c-Abl also inhibits the ability of DNA-PK to form a complex with DNA (5). c-Abl, like Ku, associates with DNA-PK_cs near the kinase homology region (29). c-Abl phosphorylates DNA-PK_cs in the C-terminal region and thereby dissociates DNA-PK_cs from the Ku-DNA complex (29). Otherwise, little is known about the regulation of DNA-PK activity.

The findings that c-Abl forms a nuclear complex with Lyn (10) and that c-Abl interacts with DNA-PK prompted studies on a potential interaction between Lyn and DNA-PK. The present results demonstrate that Lyn binds directly to DNA-PK. We also show that Lyn phosphorylates DNA-PK_cs and inhibits DNA-PK_cs activity.

	MATERIALS AND METHODS

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Cell Culture-- U-937 monoblastic leukemia cells (ATCC, Rockville, MD) were grown in RPMI 1640 media containing 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 mg/ml streptomycin, and 2 mM L-glutamine. Irradiation was performed at room temperature using a Gammacell-1000 (Atomic Energy of Canada, Ottawa, ON, Canada) under aerobic conditions with ¹³⁷Cs source emitting at a fixed dose rate of 0.76 gray/min.

Immunoprecipitation and Immunoblot Analysis-- Preparation of cell lysates and immunoprecipitations were performed as described (3). Soluble proteins were incubated with anti-Lyn (Upstate Biotechnology Inc., Lake Placid, NY) or anti-DNA-PK (Upstate Biotechnology Inc.) for 1 h and precipitated with protein A-Sepharose for an additional 1 h. The resulting immune complexes were washed three times with lysis buffer, separated by electrophoresis in SDS-polyacrylamide gels, and transferred to nitrocellulose filters. The residual binding sites were blocked by incubating the filters in 5% dry milk in phosphate-buffered saline and 0.05% Tween 20 for 1 h at room temperature followed by a 1-h incubation with anti-DNA-PK or anti-Lyn antibodies. The antigen-antibody complexes were visualized by enhanced chemiluminescence (ECL detection system, Amersham Pharmacia Biotech). Signal intensities were determined by densitometeric analysis (UltroScan; LKB, Brommer, Sweden).

Production of GST-Lyn Fusion Protein-- The pGEX plasmid encoding a GST-Lyn (amino acids 1-243) fusion protein (30) was transfected into Escherichia coli DH5, and the fusion protein was prepared by inducing log phase cells with isopropyl--D-thiogalactopyranoside. The cell pellets were lysed by sonication. The fusion proteins were purified by affinity chromatography using glutathione-Sepharose beads (Amersham Pharmacia Biotech) as described (30) and equilibrated in lysis buffer.

Fusion Protein Binding Assays and Immunoblotting-- Total cell lysates were incubated with 5 µg of immobilized GST or the indicated GST fusion proteins for 2 h at 4 °C (31). Protein complexes were washed three times with lysis buffer and boiled for 5 min in SDS sample buffer. The complexes were separated by electrophoresis in 5% SDS-polyacrylamide gels and then transferred to nitrocellulose paper. After blocking in 5% dry milk in phosphate-buffered saline and 0.05% Tween 20 for 1 h at room temperature, the filters were incubated for 1 h with anti-DNA-PK antibody and analyzed as described above.

In Vitro Transcription/Translation of DNA-PK Fragments-- DNA-PK_cs polypeptides were prepared using a coupled in vitro transcription/translation method (Promega) with templates generated from the DNA-PK_cs cDNA by polymerase chain reaction as described (29). Lyn binding to in vitro translated DNA-PK_cs polypeptides was assayed by incubating GST-Lyn SH3 (5 µg) in 20 µl of MB (10 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, pepstatin, and aprotinin) with equal amounts of each in vitro translated [³⁵S]DNA-PK_cs product for 1-2 h at 4 °C. A separate incubation of the [³⁵S]DNA-PK_cs product with GST was used as a negative control. The beads were washed four times in 1 ml of MB at 4 °C, and proteins were eluted by boiling in 2× SDS sample buffer (100 mM Tris-HCl, pH 7.0, 4% SDS, 720 nM 2-mercaptoethanol, 5 mg/ml bromphenol blue). Samples were analyzed by SDS-PAGE and autoradiography.

Direct Interaction of DNA-PK_cs with Lyn-- Purified DNA-PK (Promega) was incubated with purified Lyn (Upstate Biotechnology Inc.) in lysis buffer for 1 h at 4 °C. After incubation, proteins were subjected to immunoprecipitation with anti-Lyn or PIRS, and the precipitates were analyzed by immunoblotting with anti-DNA-PK antibody.

In Vitro Phosphorylation of DNA-PK by Lyn-- Purified DNA-PK (Promega, 0.5 µg) was incubated in the absence of DNA in kinase buffer containing [-³²P]ATP with purified kinase-active Lyn (Upstate Biotechnology Inc.) for 20 min at 30 °C. The reaction was terminated by the addition of SDS-PAGE sample buffer, and reaction products were analyzed by SDS-PAGE and autoradiography. In vitro kinase reactions containing active or heat-inactivated (HI) Lyn and DNA-PK_cs were also performed with cold ATP. Proteins were separated by SDS-PAGE, transferred to nitrocellulose filters, and analyzed by immunoblotting with anti-P-Tyr (Upstate Biotechnology Inc.). Purified Ku (provided by Dr. Lees-Miller; 0.5 µg) was incubated in kinase buffer containing [-³²P]ATP and purified kinase-active Lyn for 20 min at 30 °C. The reaction was terminated by the addition of SDS-PAGE sample buffer, and reaction products were analyzed by SDS-PAGE and autoradiography.

Dissociation of DNA-PK from DNA by Lyn-- DNA-PK/Ku (1 µg, Promega) was incubated with double-stranded DNA-cellulose beads (15 µg; U. S. Biochemical Corp.) in kinase buffer (25 mM HEPES, pH 7.4, 75 mM KCl, 10 mM MgCl₂, 1 mM dithiothreitol, 0.2 mM EGTA, 0.1 mM EDTA) for 30 min at room temperature. The DNA-cellulose beads were then washed and resuspended in kinase buffer. Kinase reactions containing beads, 100 µM ATP and active Lyn (Upstate Biotechnology Inc.), HI Lyn, or MEK1 (Upstate Biotechnology Inc.) kinases were incubated for 15 min at 30 °C. To ensure that phosphorylation was not due to DNA-PK, 20 µM wortmannin (Sigma) was added to inhibit DNA-PK activity (5). The supernatant fraction was obtained by sedimentation of the beads. Following washing of the beads with kinase buffer, the beads and supernatant fraction were boiled in SDS sample buffer. Proteins were separated by 5% or 10% SDS-PAGE and analyzed by immunoblotting with anti-DNA-PK or anti-Ku antibodies. Signal intensities were determined by densitometric analysis (UltroScan).

Inactivation of DNA-PK by Phosphorylation with Lyn-- Purified DNA-PK was incubated with purified kinase-active Lyn or HI Lyn in kinase buffer containing [³²P]ATP for 15 min at 30 °C. The reactions containing the phosphorylated DNA-PK were then incubated with GST-p53 and [³²P]ATP in KB for an additional 15 min at 30 °C. As controls, purified Lyn was also incubated with GST-p53 in the presence and absence of DNA. The kinase reactions were stopped by boiling in 2× SDS sample buffer. Eluted proteins were analyzed by SDS-PAGE and autoradiography.

	RESULTS AND DISCUSSION

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To determine whether nuclear Lyn associates with DNA-PK, we subjected anti-Lyn immunoprecipitates to immunoblot analysis with anti-DNA-PK antibodies. Whereas immunoprecipitates obtained with PIRS had no detectable DNA-PK_cs, immunoprecipitation with the anti-Lyn antibody revealed the presence of complexes containing DNA-PK_cs (Fig. 1A). To evaluate the stoichiometry of interaction between DNA-PK_cs and Lyn, we subjected U-937 cell lysates to immunoprecipitation with anti-Lyn and analyzed the precipitates by immunoblotting with anti-DNA-PK. Signal intensities from before and after anti-Lyn immunoprecipitation were compared by laser densitometric scanning. The results demonstrate that approximately 25% of DNA-PK_cs is associated with Lyn (data not shown). Because exposure of cells to IR is associated with activation of both Lyn (11) and DNA-PK (24), we also investigated whether IR affects the interaction between these proteins. IR treatment was associated with a reproducible increase in the association of Lyn with DNA-PK_cs to some extent (Fig. 1B). To confirm binding of Lyn and DNA-PK_cs, cell lysates were incubated with a GST fusion protein prepared from the 1-243 amino acid fragment of Lyn that includes a unique N-terminal region, Src homology 3 (SH3) and SH2 domains but not kinase domains (30) (Fig. 1C). Adsorbates obtained with GST-Lyn 1-243 but not with GST demonstrated binding of DNA-PK_cs (Fig. 1D and data not shown). Adsorbates obtained with GST-Lyn 1-131 and GST-Lyn 27-131 but not with GST-Lyn 131-243 also revealed specific binding of DNA-PK_cs to the Lyn SH3 domain (Fig. 1D). These findings indicate that the SH3 domain of Lyn contributes to the association with DNA-PK.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Association of DNA-PK and Lyn. A, lysates from U-937 cells were subjected to immunoprecipitation with PIRS or anti-Lyn. Immunoprecipitates were analyzed by immunoblotting with anti-DNA-PK. Cell lysate was also subjected to immunoprecipitations with anti-DNA-PK as a positive control. B, U-937 cells were treated with 20-gray IR and harvested at the indicated times. Lysates were then subjected to immunoprecipitation with anti-Lyn. Lysate was also used directly as a positive control. Immunoprecipitates were analyzed by immunoblotting with anti-DNA-PK. C, schematic diagrams of various Lyn constructions. D, U-937 cell lysate was incubated with GST-Lyn 131-243, GST-Lyn 1-243, GST-Lyn 1-131, or GST-Lyn 27-131 fusion proteins. The adsorbates were analyzed by immunoblotting with anti-DNA-PK. IB, immunoblot.

To assess whether Lyn binds directly to DNA-PK_cs, we incubated purified DNA-PK_cs with Lyn. Anti-Lyn immunoprecipitates were analyzed by immunoblotting with anti-DNA-PK. In contrast to PIRS, immunoprecipitation with anti-Lyn revealed the presence of DNA-PK_cs (Fig. 2A). These findings support a direct interaction between DNA-PK_cs and Lyn. To assess where Lyn binds to DNA-PK_cs, we prepared fragments of DNA-PK_cs (Fig. 2B) by in vitro transcription/translation (29). Equal quantities of the DNA-PK_cs in vitro translation products were incubated with GST-Lyn 1-243 bound to glutathione-Sepharose beads. Analysis of the adsorbates by autoradiography demonstrated binding of Lyn to DNA-PK_cs fragments 8, 11, and 14 (data not shown). These results support direct binding of Lyn to a leucine zipper region in DNA-PK_cs (amino acids 1503-1538). Various fragments of DNA-PKcs were also studied for binding to the Lyn SH3 domain. The Lyn SH3 domain binds to proline-rich sequences with the consensus XPPXXPX. Consistent with the presence of such a sequence (REFPPGTPRFNN, amino acids 1744-1755; fragments 11 and 14) in DNA-PK_cs, the results of Lyn SH3 binding assays with in vitro translated DNA-PK_cs fragments 11 and 14 confirmed a direct interaction (Fig. 2, C and D, and data not shown). By contrast, there was no apparent binding of Lyn SH3 to other DNA-PK_cs fragments (Fig. 2B). Collectively, these studies indicate that the association between Lyn and DNA-PK_cs occurs by direct interaction of the Lyn SH3 domain with DNA-PK_cs amino acids 1520-1976.

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Direct interaction between Lyn and DNA-PKcs. A, purified DNA-PK was incubated with Lyn in lysis buffer for 1 h at 4 °C, and anti-Lyn immunoprecipitates were analyzed by immunoblotting with anti-DNA-PK. PIRS was used as a negative control. B, protein fragments from various regions of the DNA-PKcs. C, GST Lyn 1-131 bound to glutathione-Sepharose was incubated with in vitro translated fragments of [35S]DNA-PKcs. Following washing, samples were separated in 10% SDS-PAGE gels and analyzed by autoradiography. D, GST-Lyn 1-131 or GST bound to glutathione-Sepharose was mixed with [35S]DNA-PKcs fragments 11 or 14. Following washing, the bound proteins were analyzed by SDS-PAGE and autoradiography. Arrows indicate specific DNA-PKcs polypeptides. IB, immunoblot.

To determine whether Lyn phosphorylates DNA-PK_cs, we incubated purified DNA-PK with active or HI Lyn. There was no detectable phosphorylation of DNA-PK_cs in the presence of HI Lyn (data not shown). By contrast, kinase reactions that included kinase-active Lyn demonstrated phosphorylation of DNA-PK_cs (Fig. 3A). Lyn phosphorylation of DNA-PK_cs did not require DNA-bound DNA-PK. Also, As a positive control, DNA-PK was incubated with DNA beads to show autophosphorylation of DNA-PK_cs (Fig. 3A). To confirm Lyn-dependent phosphorylation of DNA-PK_cs, we incubated purified DNA-PK_cs with active or HI Lyn in kinase buffer containing cold ATP. The phosphorylated products were analyzed by immunoblotting with anti-P-Tyr. The results demonstrate Lyn-dependent tyrosine phosphorylation of DNA-PK_cs (Fig. 3B). DNA-PK_cs forms a complex with the 70- and 80-kDa Ku heterodimer (26, 32), and thus we also incubated Lyn with purified Ku in a kinase reaction. In contrast to DNA-PK_cs, there was no detectable phosphorylation of Ku (Fig. 3C). These findings indicate that Lyn phosphorylates DNA-PK_cs and not Ku.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Lyn phosphorylates DNA-PKcs in vitro. A, purified Lyn kinase was incubated with purified DNA-PK/Ku in the presence of [32P]ATP for 15 min at 30 °C. DNA-PK/Ku complexes were also incubated with DNA in the presence of [32P]ATP for 15 min at 30 °C as a positive control for DNA-PK phosphorylation. In vitro kinase reactions were analyzed by SDS-PAGE and autoradiography. B, purified Lyn kinase was incubated with purified DNA-PK/Ku in the presence of cold ATP for 15 min at 30 °C. DNA-PK/Ku complexes were also incubated with DNA in the presence of cold ATP for 15 min at 30 °C as a control for DNA-PK autophosphorylation. In vitro kinase reactions were analyzed by SDS-PAGE and immunoblotting with anti-P-Tyr antibody. C, purified Lyn was incubated with purified Ku in the presence of [32P]ATP for 15 min at 30 °C. Purified DNA-PK/Ku was incubated with DNA beads as a positive control. In vitro kinase reactions were analyzed by SDS-PAGE and autoradiography. Sup., supernatant.

The activation of DNA-PK by binding of DNA-PK_cs to Ku/DNA is associated with phosphorylation of p53 (19). Therefore to assess the effect of Lyn on DNA-PK activity, we incubated purified DNA-PK_cs/Ku/DNA complexes with active or inactive Lyn and assessed DNA-PK_cs activity using GST-p53 as a substrate. In the presence of DNA, DNA-PK_cs/Ku phosphorylated GST-p53 (Fig. 4A, lane 1). By contrast, addition of either active or inactive Lyn to the reaction inhibited GST-p53 phosphorylation (Fig. 4A, lanes 2 and 3). In the absence of DNA-PK, Lyn had little effect on phosphorylation of GST-p53 (Fig. 4A, lane 4). These findings indicate that the direct interaction of Lyn with DNA-PK_cs, and not necessarily the Lyn kinase function, contributes to the inactivation of DNA-PK_cs. To further assess the functional significance of the interaction of Lyn and DNA-PK, the DNA-PK/Ku complex was bound to DNA beads and incubated with active or HI Lyn. The reactions included wortmannin to inhibit DNA-PK_cs autophosphorylation and thereby inhibit autodissociation from DNA (28). Incubation with kinase-active or kinase-inactive Lyn resulted in release of DNA-PK_cs from the beads into the supernatant (Fig. 4B). By contrast, addition of the MEK1 serine/threonine kinase had no detectable effect on release of DNA-PK_cs from DNA beads (Fig. 4B). Whereas Lyn phosphorylates DNA-PK_cs, but not Ku, we also asked whether Lyn affects the interaction between Ku and DNA. In the presence of Lyn, most of the Ku remained associated with the DNA beads (Fig. 4C). Similar results were obtained with the MEK1 kinase (Fig. 4C). DNA-PK_cs released from Ku/DNA in the presence of Lyn as compared with release of the DNA-PK_cs/Ku complex from DNA was assessed by immunoblot analysis of proteins in the supernatant and bound to the beads. The results demonstrate that approximately 40% of DNA-PK_cs is released from Ku in the presence of Lyn (Fig. 4D). By contrast, only 2-4% of DNA-PK_cs/Ku complexes were released from the DNA beads in the presence of Lyn (Fig. 4D). These findings demonstrate that interaction of Lyn and DNA-PK_cs results in dissociation of DNA-PK_cs and Ku, and thereby directs inhibition of DNA-PK_cs activity.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Lyn inhibits DNA-PKcs activity. A, purified DNA-PK/Ku/DNA complexes were incubated with buffer (lane 1), active Lyn kinase (lane 2), or inactive Lyn (lane 3) in the presence of [32P]ATP for 15 min at 30 °C. GST-p53 fusion protein was then added, and the reactions were incubated in the presence of [32P]ATP for an additional 15 min at 30 °C. Purified Lyn was incubated with GST-p53 in the absence of DNA-PK/Ku/DNA complexes (lane 4). The reactions were stopped by the addition of SDS sample buffer and analyzed by SDS-PAGE and autoradiography. B, purified DNA-PK/Ku was incubated with DNA beads. The beads were washed and resuspended in kinase buffer. Kinase reactions containing beads, 20 µM wortmannin, ATP were incubated with active or HI Lyn for 15 min at 30 °C. MEK1 kinase was used as a negative control. The supernatant fraction was obtained by sedimentation of the beads. The beads and supernatant fractions were boiled in SDS sample buffer. Proteins were separated by 5% SDS-PAGE and analyzed by immunoblotting with anti-DNA-PK. C, purified DNA-PK/Ku was incubated with DNA beads. The beads were washed and resuspended in kinase buffer. Kinase reactions containing beads, 20 µM wortmannin, ATP, and purified Lyn or MEK1 kinases were incubated for 15 min at 30 °C. The supernatant fraction was obtained by sedimentation of the beads. The beads and supernatant fractions were boiled in SDS sample buffer. Proteins were separated by 10% SDS-PAGE and analyzed by immunoblotting with anti-Ku. D, the percentage release of DNA-PKcs from Ku/DNA and DNA-PKcs/Ku complexes from DNA are expressed as the means ± S.D. of three independent experiments. Black bars, DNA-PKcs released from Ku/DNA; hatched bars, DNA-PKcs/Ku complexes released from DNA. Sup., supernatant.

DNA-PK is essential in the repair of DNA double-stranded breaks that form in irradiated cells (20, 33, 34). Autophosphorylation inactivates DNA-PK by a mechanism in which DNA-PK_cs dissociates from Ku (28). Other studies have shown that c-Abl negatively regulates DNA-PK in the response to DNA damage (5). The present studies demonstrate that DNA-PK is also regulated by Lyn. DNA-PK constitutively associates with Lyn by direct binding of the Lyn SH3 domain to an internal region of DNA-PK_cs that includes a leucine zipper. Lyn also phosphorylates DNA-PK_cs. The in vitro findings indicate that the direct binding of Lyn to DNA-PK_cs is sufficient to inhibit DNA-PK_cs activity. Thus, constitutive binding of Lyn and DNA-PK_cs could regulate the accessibility of certain pools of DNA-PK_cs for interaction with Ku/DNA complexes. Lyn-mediated phosphorylation of DNA-PK_cs represent another level of DNA-PK_cs regulation. These results are in concert with the demonstration that the interaction between DNA-PK_cs and Lyn induces the dissociation of DNA-PK_cs from the Ku/DNA complex and thereby inhibits DNA-PK_cs activity. The activation of Lyn by IR and other DNA damaging agents contributes to the down-regulation of Cdc2 (13-16), indicating that Lyn is an effector of cell cycle progression in the response to DNA damage. The present findings support a function for Lyn in the regulation of DNA repair. Accordingly, interactions between Lyn and DNA-PK_cs may play a role in releasing DNA-PK_cs from Ku/DNA complexes after repair to permit relocation at new sites of DNA damage.