Protein kinase activity of Tel1p and Mec1p, two Saccharomyces cerevisiae proteins related to the human ATM protein kinase

Abstract

The Saccharomyces cerevisiae proteins Tel1p and Mec1p are involved in telomere length regulation and cellular responses to DNA damage. The closest relative of these proteins is the human Ataxia Telangiectasia Mutated (ATM) protein, a wortmannin-sensitive protein kinase that primarily phosphorylates serines in an SQ motif. We constructed yeast strains containing functional epitope-tagged versions of Tel1p and Mec1p. We showed that immunoprecipitated Tel1p and Mec1p were capable of in vitro phosphorylation of the mammalian protein PHAS-I (Phosphorylated Heat and Acid Stable protein). These activities are sensitive to wortmannin. Tel1p phosphorylates serine in an SQ motif in PHAS-I. Mutations in the kinase domains of Tel1p and Mec1p result in loss of in vitro kinase activity and the in vivo phenotypes associated with the null tel1 and mec1 mutations.

Figure 1

Kinase activities of Tel1p and Mec1p. (a) Kinase activity of immunoprecipitated HA-tagged Tel1p on PHAS-I. Products of a kinase reaction using labeled ATP were analyzed by SDS/PAGE. Strains from which immunoprecipitates were derived: lane 1, JMY48 (high-copy-number plasmid with TEL1-HA); lane 2, mock immunoprecipitate (no anti-HA antibody) from JMY48; lane 3, JMY103 [high-copy number plasmid with tel1-HA(KD)]; lane 4, KRY22 (TEL1-HA in single chromosomal copy); and lane 5, JMY303–2a (tel1 strain, no epitope-tagged Tel1p). (b) Kinase activity of immunoprecipitated HA-tagged Mec1p on PHAS-I. Strains from which immunoprecipitates were derived: lane 1, JMY312–8c (tel1 sml1∷HIS3 MEC1-HA); lane 2, JMY313–9a [tel1 mec1-HA(KD) sml1∷HIS3]; and lane 3, mock immunoprecipitate (no HA-antibody) from JMY312–8c. (c) Western analysis of HA-tagged Tel1p and Mec1p proteins. Immunoprecipitated HA-tagged Tel1p or Mec1p were examined by Western analysis using HA.11 antibody and a secondary antibody-horseradish peroxidase conjugate. Lane 1, JMY48 (high-copy number plasmid with TEL1-HA); lane 2, JMY103 [high-copy number plasmid with tel1-HA(KD)]; lane 3, JMY312–8c (MEC1-HA); and lane 4, JMY313–9a [mec1-HA(KD)]. Wt, wild type; kd, kinase dead.

Figure 2

Wortmannin sensitivity of the Tel1p and Mec1p kinases. Immunoprecipitates from strains with either TEL1-HA (JMY48) or MEC1-HA (JMY312–8c) were treated with wortmannin (details in Materials and Methods) before kinase reactions were performed. Activities are normalized to a value of 1 for immunoprecipitates incubated in the absence of wortmannin. Tel1p and Mec1p activities are represented by ● and □, respectively. Error bars indicate standard deviations (total of three experiments).

Figure 3

Requirement of 20-aa “tag” (MGSSHHHHHHSSGLVPRGSH) for PHAS-I to function as a substrate for the Tel1p kinase. (a) PHAS-I was phosphorylated by an immunoprecipitate derived from a strain with TEL1-HA (JMY48); the final two washes of the immunoprecipitate were done in the absence of PMSF. A portion of the sample was treated with thrombin (Roche Molecular Biochemicals) to remove the N-terminal 17 aa; both the untreated (lane 1) and treated (lane 2) samples were examined by gel electrophoresis. (b) Kinase reactions with immunoprecipitated Tel1p were done with two samples. One sample (lane 1) contained untreated PHAS-I. The sample in lane 2 was treated with thrombin before performing the kinase reaction. Thrombin was inactivated by addition of PMSF.

Figure 4

Phenotypes of strains with mutations in the kinase domains of Tel1p and Mec1p. (a) Assay for telomere length. DNA was isolated from strains of various genotypes and treated with PstI. The resulting fragments were separated by gel electrophoresis and hybridized to a telomeric-specific probe as described in Materials and Methods. The dispersed band near the bottom of the gel represents the terminal-DNA fragments of chromosomes with Y′-containing telomeres. The strain name and genotype (indicated in parentheses) in each lane are: lanes 1 and 5, W303a (wild-type); lane 2, JMY303–2a (tel1); lanes 3 and 4, JMY103 [tel1 + plasmid-borne TEL1-HA(KD)]; lane 6, JMY73 (sml1); lane 7, JMY314–3d [mec1-HA(KD) sml1 rad5]; and lane 8, JMY313–9a [mec1-HA(KD) sml1 tel1∷ura3]. (b) Assay for growth. Spore colonies derived from the diploids JMY313 and JMY314 were streaked onto plates containing rich growth medium and incubated at 30^o for 2 days. The two tel1 mec1-HA(KD) sml1 strains are JMY313–3a and JMY313–9a, and the two mec1-HA(KD) sml1 strains are JMY314–3d and JMY314–5a. (c) Assay of response to DNA-damaging agents. Serial dilutions of six strains were prepared on four plates containing rich growth medium (yeast extract/peptone/dextrose). One plate was untreated without any DNA-damaging agent; one was treated for 10 sec with UV light derived from a germicidal lamp; one contained 10 mM hydroxyurea (HU); and one contained 2 μg/ml streptonigrin. The strains used in the study (row 1 at the tops of the plates) were: row 1, W303a; row 2, W1588–4c (identical to W303a except RAD5 instead of rad5–535); row 3, JMY303–1c; row 4, JMY303–8d; row 5, JMY314–3d; and row 6, JMY314–5a (identical to JMY314–3d except RAD5). Cells were grown for 2 days at 30°C.