Fig. 4: SPA1 directly phosphorylates eIF2α in the C-terminal domain in vitro but not at Ser56. | Nature Communications

Fig. 4: SPA1 directly phosphorylates eIF2α in the C-terminal domain in vitro but not at Ser56.

From: The phosphorylation of carboxyl-terminal eIF2α by SPA kinases contributes to enhanced translation efficiency during photomorphogenesis

Fig. 4

a An in vitro kinase assay showed that SPA1 protein purified from P. pastoris phosphorylates eIF2α (autoradiogram on the right). The left figure showed the protein levels in a Coomassie blue staining gel. The conserved amino acid mutation Ser56 to alanine (S56A) did not reduce the phosphorylation signal of eIF2α. Three biological repeats of data showed the same results. b, c The in vitro kinase assay showed that SPA1 protein purified from P. pastoris phosphorylates C-terminal eIF2α.1 and eIF2α.2 (autoradiogram on the right), respectively. The left figure showed the protein levels in a Coomassie blue staining gel. N-terminal (N), middle (M), and C-terminal (C) domains of eIF2α were shown. A conserved amino acid mutation on the SPA1 kinase domain (mSPA1) reduced the phosphorylation activity of SPA1 on eIF2α. Three biological repeats of data showed the same results. d A table showing the relative phosphorylation signal intensities of autoradiogram normalized to protein contents detected by immunoblotting as shown in Supplementary Fig. 8. The ratio of the phosphorylation of full-length eIF2α was set to 1 for analysis.

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