Conexión de la ruta de proteinquinasa C con la respuesta a estrés genotóxico

El mantenimiento de la integridad genómica es un aspecto crucial para la supervivencia celular. El checkpoint de integridad del DNA es el mecanismo de vigilancia encargado de detectar la presencia de daño en el DNA y poner en marcha una respuesta celular para mantener la estabilidad genómica. Los componentes centrales del checkpoint son las quinasas sensoras ATM y ATR (Tel1 y Mec1 en S. cerevisiae) y CHK1 Y CHK2 (Chk1 y Rad53 en levadura). La correcta activación del checkpoint depende de la actividad de proteína quinasa C (PKC), tanto en células de levadura como de mamíferos. En este trabajo se ha profundizado en la relación entre PKC y la maquinaria del checkpoint de integridad del DNA, así como en la regulación de la propia PKC en condiciones de estrés genotóxico. Se han caracterizado los determinantes estructurales de Pkc1 de levadura y las isoformas de mamíferos PKCδ y PKCθ que les permiten llevar a cabo su función en la activación de la respuesta a daño en el DNA. Asimismo, se ha visto que existe una regulación espacial de Pkc1, PKCδ y PKCθ en respuesta a estrés genotóxico y que se produce una acumulación nuclear tras la inducción de daño en el DNA. Por otra parte, se ha abordado la identificación de mediadores de PKC en su función en la activación del checkpoint de integridad del DNA, concluyendo que en ausencia de Pkc1 se produce una ineficiente activación de Mec1 que afectaría a la activación de Rad53, comprometiendo la respuesta mediada por el checkpoint de integridad del DNA.The maintenance of genomic integrity is a crucial aspect for cell survival. The DNA integrity checkpoint is the surveillance mechanism responsible for detecting the presence of DNA damage and launching a cellular response to maintain genomic stability. The central components of the checkpoint are the ATM and ATR sensor kinases (Tel1 and Mec1 in S. cerevisiae) and CHK1 and CHK2 (Chk1 and Rad53 in yeast). The correct activation of the checkpoint depends on the activity of protein kinase C (PKC), both in yeast and mammalian cells. In this work, the relationship between PKC and the DNA integrity checkpoint machinery has been deepened, as well as the regulation of PKC itself in conditions of genotoxic stress. The structural determinants of yeast Pkc1 and the mammalian PKCδ and PKCθ isoforms that allow them to carry out their function in the activation of the response to DNA damage have been characterized. Likewise, it has been seen that there is a spatial regulation of Pkc1, PKCδ and PKCθ in response to genotoxic stress and that a nuclear accumulation occurs after the induction of DNA damage. On the other hand, the identification of PKC mediators in their function in the activation of the DNA integrity checkpoint has been addressed, concluding that in the absence of Pkc1 there is an inefficient activation of Mec1 that would affect the activation of Rad53, compromising the response mediated by the DNA integrity checkpoint

Chimeric proteins tagged with specific 3xHA cassettes may present instability and functional problems.

Epitope-tagging of proteins has become a widespread technique for the analysis of protein function, protein interactions and protein localization among others. Tagging of genes by chromosomal integration of PCR amplified cassettes is a widely used and fast method to label proteins in vivo. Different systems have been developed during years in the yeast Saccharomyces cerevisiae. In the present study, we analysed systematically a set of yeast proteins that were fused to different tags. Analysis of the tagged proteins revealed an unexpected general effect on protein level when some specific tagging module was used. This was due in all cases to a destabilization of the proteins and caused a reduced protein activity in the cell that was only apparent in particular conditions. Therefore, an extremely cautious approach is required when using this strategy

Analysis of the cellular content of proteins labelled with different tags.

<p>Cells of the wild-type (W303-1a), <i>DDC1-3xHA</i> (JCY1701), <i>DDC1-6xHA</i> (JCY1825), <i>DDC1-GFP</i> (JCY1661), <i>DDC1-myc</i> (JCY1887), <i>CLN2-3xHA</i> (JCY1357), <i>CLN2-6XHA</i> (JCY1830), <i>CLN2-GFP</i> (JCY1888), <i>CLN2-myc</i> (JCY1890), <i>PKC1-3xHA</i> (JCY2033), <i>PKC1-6XHA</i> (1891), <i>PKC1-GFP</i> (JCY1511), <i>PKC1-myc</i> (JCY1916), <i>RAD53-3xHA</i> (JCY1905), <i>RAD53-6XHA</i> (JCY1901), <i>RAD53-GFP</i> (JCY1903) and <i>RAD53-myc</i> (JCY1907) strains were grown in YPD. Protein level was determined by western blot using anti-Ddc1, anti-Cln2, anti-Pkc1 and anti-Rad53 antibodies. Cdc28 recognized by the anti-PSTAIRE antibody is shown as a loading control.</p

Analysis of the effect of the spacer sequence in 3xHA-tagging modules.

<p>(A) Schematic representation of Ddc1-3xHA, Ddc1-GFP and Ddc1-myc including the spacer sequence between the Ddc1 protein and the tag. (B) Cells of the wild-type (W303-1a), <i>DDC1-3xHA</i> (JCY1701) and <i>DDC1-3xHA</i>^<i>ΔIF</i> (JCY2063) strains were grown in YPD. Ddc1 protein level in cell extracts was determined by western blot using a specific anti-Ddc1 antibody. The ponceau staining of the membrane is shown as a loading control. (C) Exponentially growing cultures of the wild-type (W303-1a), <i>DDC1-3xHA</i> (JCY1701) and <i>DDC1-3xHA</i>^<i>ΔIF</i> (JCY2063) strains were incubated in the presence of 100 μg/mL cycloheximide. Ddc1 protein level was analysed at the indicated time after the addition of cycloheximide by western blot using an anti-Ddc1 antibody. Cdc28 recognized by the anti-PSTAIRE antibody is shown as a loading control. (D) 10-fold serial dilutions from exponentially growing cultures of wild-type (W303-1a), <i>DDC1-3xHA</i> (JCY1701) and <i>DDC1-3xHA</i>^<i>ΔIF</i> (JCY2063) strains were spotted onto YPD medium and exposed to UV radiation (40 J/m²). Plates were incubated at 25° for 4 days. (E) Whi7 was tagged with 3xHA using either the transforming modules described in Longtine <i>et al</i>. (JCY1544, lane 1) or an alternative cassette (JCY1728, lane 2). Whi7 protein level in cell extracts was determined by western blot using an anti-HA antibody. Note that Whi7 migrates in SDS-PAGE as multiple bands, which correspond to distinct phosphorylated states. The ponceau staining of the membrane is shown as a loading control.</p

Analysis of the cellular content of different 3xHA tagged proteins.

<p>Cells of the wild-type (W303-1a), <i>DDC1-3xHA</i> (JCY1701), <i>CLN2-3xHA</i> (JCY1357), <i>PKC1-3xHA</i> (JCY2033), <i>SLT2-3xHA</i> (JCY411), <i>RAD53-3xHA</i> (JCY1905) and <i>SWI5-3xHA</i> (JCY487) strains were grown in YPD. Ddc1, Cln2, Pkc1, Slt2, Rad53 and Swi5 protein level in cell extracts was determined by western blot using specific anti-Ddc1, anti-Cln2, anti-Pkc1, anti-Slt2, anti-Rad53, anti-Swi5 or anti-HA antibodies. The ponceau staining of the membrane is shown as a loading control.</p

Analysis of the stability of proteins labelled with different tags.

<p>(A) Exponentially growing cultures of the wild-type (W303-1a), <i>DDC1-3xHA</i> (JCY1701), <i>DDC1-6xHA</i> (JCY1825), <i>DDC1-GFP</i> (JCY1661) and <i>DDC1-myc</i> (JCY1887) strains were incubated in the presence of 100 μg/mL cycloheximide. Ddc1 protein level was analysed at the indicated times after the addition of cycloheximide by western blot. Cdc28 recognized by the anti-PSTAIRE antibody is shown as a loading control. (B) Cln2 protein stability was analysed in wild-type (W303-1a), <i>CLN2-3xHA</i> (JCY1357), <i>CLN2-6XHA</i> (JCY1830), <i>CLN2-GFP</i> (JCY1888) and <i>CLN2-myc</i> (JCY1890) cells as described in A. (C) Pkc1 protein stability was analysed in wild-type (W303-1a), <i>PKC1-3xHA</i> (JCY2033), <i>PKC1-6XHA</i> (1891), <i>PKC1-GFP</i> (JCY1511) and <i>PKC1-myc</i> (JCY1916) cells as described in A. (D) Rad53 protein stability was analysed in wild-type (W303-1a), <i>RAD53-3xHA</i> (JCY1905), <i>RAD53-6XHA</i> (JCY1901), <i>RAD53-GFP</i> (JCY1903) and <i>RAD53-myc</i> (JCY1907) as described in A. Protein level was determined by western blot using anti-Ddc1, anti-Cln2, anti-Pkc1, anti-Rad53, anti-HA, anti-GFP or anti-myc antibodies as indicated. Cdc28 recognized by the anti-PSTAIRE antibody is shown as a loading control.</p

Yeast strains.

<p>Yeast strains.</p