In vertebrates, cofilin is regulated by pH, phosphorylation and phosphoinositides

In vertebrates, cofilin is regulated by pH, phosphorylation and phosphoinositides

bstrate connections need to arise and the balance with competing phosphatases must be tuned to generate advantageous networks that support the ever-elaborating diversity of life. Note added in proof During the process of submission the authors have learned that the DFGx specificity mutation described in this study is also observed in multiple cancer kinases including Aurora A, PKCgamma, Haspin, DDR1, ITK, TRKA, IRAK3 and BRAF, suggesting that amino acid changes that occur during evolution are resampled in cancer. It will be interesting to see if these mutations cause specificity changes in the context of oncogenesis. Materials and methods Plasmids Kinases were Cobicistat web either cloned by PCR from respective organisms or from gifts: N. crassa Ime2 was a gift from Louise Glass, ICK and MOK cDNA were gifts from Tom Sturgill and Zheng Fu, MAK cDNA was a gift from Alex Bullock, N. gruberi genomic DNA was a gift from Lillian Fritz-Laylin. Ancestral kinases were synthesized either from gBlock gene fragments or by Genscript. All plasmids were assembled by Gibson isothermal assembly, cloned in E. coli XL1-blue strains, and prepared by miniprep. The list of plasmids used in this study is presented in Supplementary file 1. Clarification of mammalian RCK family gene names The RCK family of kinase is identified by various synonyms in the literature. Therefore, to avoid confusion, the mammalian RCK kinases used in this study are: 1. ICK Mus musculus; 2. MOK Mus musculus; 3. MAK Homo sapiens. Howard et al. eLife 2014;3:e04126. DOI: 10.7554/eLife.04126 15 of 22 Research Article Biochemistry Genomics and Evolutionary Biology Yeast strains Yeast strains were generated by standard transformation and crossing protocols. Protein purification was performed from W303 strains. We initially performed meiotic experiments in SK1 strains derived from the Herskowitz collection, but later switched to SK1 strains from Angelika Amon. Both SK1 strains gave similar results but the Amon background was more consistent. All yeast strains were generated by standard LiAc transformation. SK1 and W303 strains were heat shocked at 42C for 15 and 40 min respectively. Point mutations of IME2 in the SK1 background were generated by 2-step loop-in, loop-out gene replacement technique using selection and counter-selection of the URA3 marker at the IME2 genomic locus. The list of yeast strains used in this study is presented in Supplementary file 2. Synchronous sporulation timecourses Liquid sporulation was conducted at 30C as follows: strains were thawed on YP + 3% glycerol plates overnight, then patched on YPD plates, and grown overnight. 2 ml YPD liquid cultures were inoculated from patches and grown to saturation by shaking at 30C, 250 rpm for 2123 hr. Cultures were diluted in 50 mL of YP + 1% KOAc to OD600 = 0.25 and grown overnight by shaking at 30C, 250 rpm for 1516 hr. Cells were pelleted and washed once with sterile water, then resuspended in 1% KOAc sporulation media to OD600 2.5. Sporulation cultures were PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19825521 shaken at 30C, 250 rpm and samples were collected every hour for DNA staining and flow cytometry. DNA staining for flow cytometry and imaging Cells were fixed by mixing 0.5 ml of sporulation culture with 1 ml of EtOH. Fixed cells were pelleted and resuspended in water, then sonicated on a Branson Sonifier Model 450 at 10% amplitude for 3 s to break up cell clumps. Cells were pelleted, then resuspended in 100 ml of 40 mg/ml RNase A + 0.05% NP-40 + 50 mM Tris pH 7.4, and incubated at

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