Abstract
Sporulation in the budding yeast Saccharomyces cerevisiae is a developmental program initiated in response to nutritional deprivation. Sps1, a serine/threonine kinase, is required for sporulation, but relatively little is known about the molecular mechanisms through which it regulates this process. Here we show that SPS1 encodes a bona-fide member of the GCKIII subfamily of STE20 kinases, both through phylogenetic analysis of the kinase domain and examination of its C-terminal regulatory domain. Within the regulatory domain, we find Sps1 contains an invariant ExxxPG region conserved from plant to human GCKIIIs that we call the EPG motif; we show this EPG motif is important for SPS1 function. We also find that Sps1 is phosphorylated near its N-terminus on Threonine 12, and that this phosphorylation is required for the efficient production of spores. In Sps1, Threonine 12 lies within a 14-3-3 consensus binding sequence, and we show that the S. cerevisiae 14-3-3 proteins Bmh1 and Bmh2 bind Sps1 in a Threonine 12-dependent fashion. This interaction is significant, as BMH1 and BMH2 are required during sporulation and genetically interact with SPS1 in sporulating cells. Finally, we observe that Sps1, Bmh1 and Bmh2 are present in both the nucleus and cytoplasm during sporulation. We identify a nuclear localization sequence in Sps1 at amino acids 411–415, and show that this sequence is necessary and sufficient for nuclear localization. Taken together, these data identify regions within Sps1 critical for its function and indicate that SPS1 and 14-3-3s act together to promote proper sporulation in S. cerevisiae.
Highlights
Yeast deprived of a fermentable carbon source and nitrogen undergo sporulation [1]
We noticed that when compared to human Ste20 kinases, the kinase domain of Sps1 shares,50% amino acid identity with the germinal center kinases (GCKs) (e.g., 52% identity with the human GCKIII Ysk1), but only,40% with the p21-activated kinases (PAKs) (e.g., 40% with the human Pak1)
This raised the possibility that rather than being an outlier among Ste20 kinases, Sps1 might belong to the GCK subfamily, and prompted us to revisit the evolutionary relationship of the Sps1 kinase domain to other Ste20 family members
Summary
Sporulation begins with meiosis, which results in the production of four haploid nuclei from a single diploid cell. These four nuclei are encapsulated by the prospore membrane, which acts as the template for spore wall deposition. STE20 family kinases are highly conserved from yeast to mammals and are divided into two subgroups, the p21-activated kinases (PAKs) and the germinal center kinases (GCKs) [3,4]. These two subgroups are distinguished both by the phylogenetic relationships among their kinase domains and by their domain architectures: In PAKs, the kinase domain is C-terminal to the regulatory domain, and this is reversed in GCKs [5]. Within the GCKs, the GCKIII subfamily of kinases includes the mammalian kinases MST3, MST4, and YSK1/SOK1/STK25 [3], which have been implicated in processes such as apoptosis [6] and axon outgrowth [7], and may be involved in diseases such as Alzheimer’s [8], type 2 diabetes [9], Parkinson’s disease [10], and cerebral cavernous malformations [4]
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