Characterization of ATM expression, localization, and associated DNA-dependent protein kinase activity

Abstract

Ataxia telangiectasia-mutated gene (ATM) is a 350-kDa protein whose function is defective in the autosomal recessive disorder ataxia telangiectasia (AT). Affinity-purified polyclonal antibodies were used to characterize ATM. Steady-state levels of ATM protein varied from undetectable in most AT cell lines to highly expressed in HeLa, U2OS, and normal human fibroblasts. Subcellular fractionation showed that ATM is predominantly a nuclear protein associated with the chromatin and nuclear matrix. ATM protein levels remained constant throughout the cell cycle and did not change in response to serum stimulation. Ionizing radiation had no significant effect on either the expression or distribution of ATM. ATM immunoprecipitates from HeLa cells and the human DNA-dependent protein kinase null cell line MO59J, but not from AT cells, phosphorylated the 34-kDa subunit of replication protein A (RPA) complex in a single-stranded and linear double-stranded DNA-dependent manner. Phosphorylation of p34 RPA occurred on threonine and serine residues. Phosphopeptide analysis demonstrates that the ATM-associated protein kinase phosphorylates p34 RPA on similar residues observed in vivo. The DNA-dependent protein kinase activity observed for ATM immunocomplexes, along with the association of ATM with chromatin, suggests that DNA damage can induce ATM or a stably associated protein kinase to phosphorylate proteins in the DNA damage response pathway.

Figure 1

Characterization of antibodies raised against ATM. (A) Three antibodies raised against ATM recognize a 350-kDa band in Western blots of HeLa whole-cell lysates (20 μg) that were separated on 4–12% gradient SDS-PAGE gels. (B) Each of the three ATM antibodies immunoprecipitated a 350-kDa band that is recognized by the other two antibodies on Western blots.

Figure 2

Comparison of ATM protein levels in different cell lines. (A) Whole-cell lysates of 10⁶ NHF and HeLa and U2OS cells and of 10⁷ K562, Jurkat, and HL-60 cells probed with Ab3. (B) Whole-cell lysates (20 μg) of normal human lymphocytes (NHL) and of AT3ABR, AT5ABR, AT5BI, AT2SF, AT3Be, and HeLa cells separated and probed with Ab3. Top arrow, full-length ATM; bottom arrow, truncated ATM in AT3Be.

Figure 3

Subcellular localization of ATM. (A) Irradiated and unirradiated normal human fibroblasts fractionated as described in MATERIALS AND METHODS. Protein from unfractionated cells and from the nucleus, cytoplasm, and membrane fractions of an equal number of cells was probed with Ab3. Purity of the nuclear fraction was assessed by probing the same filter for the 367-kDa nuclear matrix protein CENP-F. (B) Subnuclear localization of ATM. NHF were sequentially extracted with 1% digitonin, 100 μg/ml DNase I, 2 M NaCl, and RIPA buffer (soluble matrix). ATM was immunoprecipitated with Ab1 from each of the soluble fractions that were adjusted to identical composition to minimize differences in immunoprecipitation efficiency. The insoluble pellet was directly dissolved in SDS sample buffer and loaded as the insoluble matrix. ATM was also immunoprecipitated from an equal number of NHF to serve as a control for the efficiency of ATM recovery (Total). The filter was probed with Ab3. (C) Immunofluorescence staining with Ab3 (right) of NHF (top) and AT2SF cell lines (bottom). Nuclei were visualized by DAPI staining (left).

Figure 4

Comparison of the steady-state levels of ATM protein across the cell cycle. (A) Ab1 immunoblot of HeLa whole-cell lysates synchronized by double thymidine block and harvested at 1–24 h after release. (B) Ab1 immunoblot of K562 whole-cell lysates after separation by centrifugal elutriation. (C) Ab1 immunoblot of NHF serum starved for 24 h in 0.5% FBS and harvested at 1–24 h after stimulation with 20% FBS.

Figure 5

The effect of IR on expression of ATM protein. (A) Ab1 immunoblot of HeLa cells treated with 2 Gy of IR and harvested 1–24 h after treatment. (B) Ab1 immunoblot of HeLa cells treated with 10 Gy of IR and harvested 1–24 h after treatment. (C) Ab1 immunoblot of NHF treated with 10 Gy of IR and harvested 1–24 h after treatment.

Figure 6

Phosphorylation of p34 RPA by ATM-associated protein kinase. All kinase reactions were performed with immunoprecipitates from equal amounts of cellular protein. (A) ATM immunoprecipitated from HeLa cells with nonimmune rabbit IgG (Nonimmune IgG, lane 1), peptide-blocked Ab3 (Blocked, lane 2), or Ab3 (ATM Ab3, lane 3) and incubated with [γ-³²P]ATP, ssDNA, dsDNA, and purified RPA. (B) ATM protein kinase activity immunoprecipitated from HeLa (lanes 1 and 2), MO59K (lane 3), AT2SF (lane 4), and MO59J (lane 5) cells. (C) Lysate (50 μg) from HeLa (lane 1), AT2SF (lane 2), MO59J (lane 3), and MO59K (lane 4) cells probed with antibodies to DNA-PK (top) or ATM Ab3 (bottom). (D) ATM-associated protein kinase activity in AT2SF cells (AT) (lanes 1 and 2) or in HeLa cells (H) (lanes 3–9) with purified RPA used as a substrate (lanes 1–7). (E) Kinase reactions performed on immunoprecipitates from MO59J cells with blocked Ab3 (lane 1) and Ab3 (lanes 2 and 3) in the presence (lanes 1 and 2) or absence (lane 3) of dsDNA.

Figure 7

ATM-associated protein kinase phosphorylation of p34 RPA on serine and threonine residues. Two-dimensional phosphoamino acid analysis of phosphorylated p34 RPA is shown.

Figure 8

Phosphopeptide maps of p34 RPA phosphorylated in vitro by ATM-associated protein kinase and in vivo in response to IR. In vivo–phosphorylated p34 RPA (A) and in vitro–phosphorylated p34 RPA (B) were digested with trypsin and endoproteinase lys-C and separated by electrophoresis in pH 3.5 buffer in the first dimension and ascending chromatography in the second dimension. Numbered areas correspond to phosphopeptides that are common in the two maps; arrows in A identify peptides that are specific to the in vivo sample.