Figure 4

Localisation, not substrate specificity, distinguishes p38α from p38β. (A) FSBA-treated C2C12 cell lysate was incubated with a kinase assay buffer and p38α, p38β or no kinase as a control. Both isoforms produced similar banding patterns. The prominent bands marked with the asterisks in each of the p38α and p38β lanes represent autophosphorylation of the added recombinant kinase. (B) The quantitative values for all 158 identified p38α phosphorylations are plotted in blue on a log₂ scale, with the corresponding values for p38β shown in red. The dashed line represents the fold cutoff for accepted substrates. Four p38β phosphorylations fell appreciably below the fold cutoff (point 2 corresponds to serine 27 of the 40S ribosomal protein S27-Rps27). (C) Incubation of p38α or p38β with purified Rps27 and a kinase assay buffer. (D) Incubation of p38α or p38β with a peptide from Rps27 containing serine 27 (or mutation of serine 27 to alanine) and a kinase assay buffer. (C) and (D) demonstrate that serine 27 of Rps27 is not a specific p38α phosphorylation site. (E) FLAG-tagged p38α or p38β were transfected into C2C12 cells, and at 48 hours of differentiation immunofluorescence staining for FLAG was performed. p38α has a ubiquitous distribution, whereas p38β is found solely at the cell periphery. Cells were imaged using a Zeiss LSM 510 META confocal microscope (Carl Zeiss MicroImaging GmbH, Jena, Germany). Scale bar = 20 μm.