Ser 524 is a phosphorylation site in MUTYH and Ser 524 mutations alter 8-oxoguanine (OG): a mismatch recognition

Abstract

MUTYH-associated polyposis (MAP) is a colorectal cancer predisposition syndrome that is caused by inherited biallelic mutations in the base excision repair (BER) gene, MUTYH. MUTYH is a DNA glycosylase that removes adenine (A) misinserted opposite 8-oxo-7,8-dihydro-2'-deoxyguanosine (OG). In this work, wild type (WT) MUTYH overexpressed using a baculovirus-driven insect cell expression system (BEVS) provided significantly higher levels of enzyme compared to bacterial overexpression. The isolated MUTYH enzyme was analyzed for potential post-translational modifications using mass spectrometry. An in vivo phosphorylation site was validated at Serine 524, which is located in the C-terminal OG recognition domain within the proliferating cell nuclear antigen (PCNA) binding region. Characterization of the phosphomimetic (S524D) and phosphoablating (S524A) mutants together with the observation that Ser 524 can be phosphorylated suggest that this residue may play an important regulatory role in vivo by altering stability and OG:A mismatch affinity.

Figure 1. SDS-PAGE (left panel) and Western blot (right panel) analysis of WT MUTYH (I)

Lane 1: Control E. coli MBP MutY, Lane 2: MUTYH (E), Lane 3: Marker, Lane 4: Uninfected control Sf9 cells, Lane 5: Pellet fraction from whole cell lysates infected with MUTYH baculovirus, Lane 6: Supernatant from whole cell lysate infected with WT bvMBP MUTYH baculovirus. Left Panel: Samples were run on a 12 % SDS-PAGE gel and stained with Sypro orange. The arrow in lane 4 marks the missing band in uninfected cells, which appears in the infected cells, lanes 5 and 6. The band for MUTYH (I) is present between the 97–116 kDa markers thus falling in the expected region (~102 kDa). Right panel: A second SDS-PAGE gel was run with identical samples and then western blotted with an anti-MBP antibody.

Figure 2. Sequence coverage of MUTYH obtained by LC-MS/MS analysis

Sequence underlined is the peptide coverage obtained. Peptide identified that upon validation identified Ser 524 as phosphorylated site is shown boxed.

Figure 3. Mass Spectrometry Analysis leading to validation of a phosphorylation of Ser 524 of MUTYH

(A) List of potential phosphorylated peptides based on masses observed in LC-MS/MS analysis. Phosphopeptide 1 shown in bold was validated as the correct site of phosphorylation. The lower case p indicates the following serine is phosphorylated. (B) Product ion spectra of the [M + 2H]²⁺ ion from SHIpSTDAHSLNSAAQ acquired by ESI-FTICR. (C) Fragmentation table for SHIpSTDAHSLNSAAQ. Orange and blue highlighted m/z values indicate the presence of standard b- and y-type fragment ions, respectively, whereas green-highlighted product ions are indicative of special losses from the b and y ions.

Figure 4. Adenine Glycosylase activity of WT MUTYH (I)

(A) A representative plot of product formation as a function of time under multiple-turnover conditions with WT MUTYH (I) [1.25 nM] on a OG:A-containing DNA duplex [20 nM] at 37 °C. The graph illustrates a biphasic behavior for MUTYH (I) with an initial burst of product formation followed by a slower steady-state phase. (B) A representative plot of product formation as a function of time under single-turnover conditions with WT MUTYH (I) [11 nM] on a OG:A-containing DNA duplex [1 nM] at 37 °C. (C) Minimal kinetic scheme for MUTYH (I). All enzyme concentrations listed are active enzyme concentrations based on active site titration experiments.

Figure 5. Adenine Glycosylase Activity of S524D and S524A MUTYH

(A) Bar graph comparing the stability of the phosphomutants with WT MUTYH (I); WT (black), S524D (gray), S524A (vertical stripes). Activity was measured by incubation of WT MUTYH (I) and phosphomutants in assay buffer. Aliquots were removed at intervals of 0, 5 and 10 min for adenine glycosylase assays under multiple-turnover conditions. The activity of MUTYH (I) without prior incubation was normalized to 1. The bars at 5 and 10 min represent the average % loss of activity on incubation with respect to the normalized data. The error is one standard deviation from the average. (B) Representative plots of product formation as a function of time under single-turnover conditions with phosphomutants S524A (closed squares) and S524D (open circles) MUTYH on a OG:A-containing DNA duplex [5 nM] at 37 °C. Rate constants determined from appropriate fitting of the data and averaged from several experiments are listed in Table 1.

Figure 6. DNA Mismatch Afffinity of WT, S524D and S524A MUTYH

Representative storage phosphor autoradiogram and graphical fitting of EMSA data for WT MUTYH bound to a 30-mer DNA duplex containing FA, 1N and THF. (A) Synthetic analogues used for EMSA studies. (B) Gel image of EMSA data for WT MUTYH bound to a 30-mer duplex with a central OG:A mispair. (C) Plot of percent WT MUTYH bound to duplex containing a central OG:FA (open circles), OG:1N (closed circles) or OG:THF (open squares) mispair. The % bound has been normalized to maximum amount bound with a given duplex. [DNA] =0.01 nM, [MUTYH] = 0.05 to 21 nM. (D) Representative graphical plot of percent WT (open circles), S524A (closed circles) and S524D (open squares) MUTYH (I), bound to a DNA duplex containing a central OG: FA^β mispair. The % bound has been normalized to maximum amount bound with a given enzyme. The K_d values from at least 4 separate experiments are listed in Table 1

Figure 7. Sequence alignment of different mammalian MutY homologues in the region corresponding to 511 to 525 in MUTYH

The alignment shows the high level of conservation of Ser 524 (indicated in bold) identified as an in vivo phosphorylation site.