Phosphorylation-Independent Regulation of Atf1-Promoted Meiotic Recombination by Stress-Activated, p38 Kinase Spc1 of Fission Yeast

Abstract

BackgroundStress-activated protein kinases regulate multiple cellular responses to a wide variety of intracellular and extracellular conditions. The conserved, multifunctional, ATF/CREB protein Atf1 (Mts1, Gad7) of fission yeast binds to CRE-like (M26) DNA sites. Atf1 is phosphorylated by the conserved, p38-family kinase Spc1 (Sty1, Phh1) and is required for many Spc1-dependent stress responses, efficient sexual differentiation, and activation of Rec12 (Spo11)-dependent meiotic recombination hotspots like ade6-M26.Methodology/Principal FindingsWe sought to define mechanisms by which Spc1 regulates Atf1 function at the ade6-M26 hotspot. The Spc1 kinase was essential for hotspot activity, but dispensable for basal recombination. Unexpectedly, a protein lacking all eleven MAPK phospho-acceptor sites and detectable phosphorylation (Atf1-11M) was fully proficient for hotspot recombination. Furthermore, tethering of Atf1 to ade6 in the chromosome by a heterologous DNA binding domain bypassed the requirement for Spc1 in promoting recombination.Conclusions/SignificanceThe Spc1 protein kinase regulates the pathway of Atf1-promoted recombination at or before the point where Atf1 binds to chromosomes, and this pathway regulation is independent of the phosphorylation status of Atf1. Since basal recombination is Spc1-independent, the principal function of the Spc1 kinase in meiotic recombination is to correctly position Atf1-promoted recombination at hotspots along chromosomes. We also propose new hypotheses on regulatory mechanisms for shared (e.g., DNA binding) and distinct (e.g., osmoregulatory vs. recombinogenic) activities of multifunctional, stress-activated protein Atf1.

A Stress-Activated, p38 Mitogen-Activated Protein Kinase–ATF/CREB Pathway Regulates Posttranscriptional, Sequence-Dependent Decay of Target RNAs

“…Cells were cultured at 32°C using rich yeast extract agar (YEA) or liquid (YEL), minimal nitrogen base agar (NBA) or liquid (NBL), or defined Edinburgh minimal medium (EMM2), supplemented with required amino acids and bases at 100 g/ml (31,32). Standard genetic methods (31), with modifications to improve the mating efficiency of partially sterile mutants (11), were used to construct new strains. The genotypes of strains were determined by a combination of phenotyping, PCR diagnostics, and restriction mapping and DNA sequence analyses of PCR products (4,31,33).…”

“…In the fission yeast Schizosaccharomyces pombe, the protein kinase Spc1 (Sty1/Phh1) is a p38-family SAPK that is part of a welldefined signal transduction pathway orthologous to p38 SAPK cascades of mammals, Drosophila, and other metazoans (2). This pathway regulates numerous cellular functions, including resistance to stress (3,4), a G 0 cell cycle checkpoint (5-7), chronological life span (8), sexual differentiation (3,6,9,10), and the positioning of meiotic recombination (4,11).…”

“…Atf1 is phosphorylated by the p38-family SAPK Spc1, and Spc1 is required for essentially all Atf1-dependent functions (e.g., see references 3, 4, 9, and 16). Paradoxically, the canonical proline-directed serine/threonine phosphoacceptor sites of Atf1 are dispensable for some functions (11,17).…”

“…The Atf1-dependent recruitment of Spc1 to promoters is thought to facilitate transcriptional elongation (13,16,19). Other Spc1-dependent functions of the heterodimer, such as activation of meiotic recombination hotspots, do not require recruitment of Spc1 to the chromosome (11). The Atf1-Pcr1-M26 protein-DNA complex induces both euchromatin and heterochromatin (7,(20)(21)(22).…”

Embryonic Development of the Ovary, Sexual Reproduction and Meiosis

“…Mutation of one base of this motive strongly reduces the hotspot activity (Myers et al 2006); protein-DNA complexes are essential for hotspot activity. The ade6-M26 hotspot (chromosomal region) of fission yeast is the most extensively characterized and best understood meiotic hotspot in eukaryotes (Gao et al 2009). Atf1-Pcr1 protein heterodimer associates with ade6-M26 hotspot.…”