Figure 5: ROCK2 activity is independent of activation loop phosphorylation.

(a) Alignment of activation loops for the ROCK subfamily of human AGC kinases. The canonical activation loop phosphorylation site of the AGC kinases is marked with a red box. The alignment includes a representative ROCK orthologue from the arthropod phylum (D. melanogaster), highlighted in blue. Proteins for which high-resolution structures exist of the unphosphorylated kinase domain with an ordered activation loop in the conformation characteristic of active eukaryotic protein kinases are indicated with a closed circle. (b) Active conformation of the ROCK2 kinase domain illustrated by the crystal structure (PDB ID: 2F2U). The ordered activation loop is highlighted in blue mesh, while the unphosphorylated canonical phosphorylation site (ROCK2 T240) is indicated in red. (c) Immunostaining of NIH3T3 ΔROCK2 cells expressing a non-phosphorylatable EGFP-ROCK2^T240A or a phosphomimetic EGFP-ROCK2^T240E. Cells show normal stress fibres indistinguishable from untransfected cells. (d) ROCK2^T240A activity against MLC2 is indistinguishable from wild-type ROCK2 in vitro. EGFP-ROCK2 was immobilized in wells of a GFP-Trap 96-well plate and subjected to an in vitro kinase assay. Immunoblot against pS¹⁹ MLC2. ROCK2^T240A activity was normalized to ROCK2^WT using EGFP fluorescence. (e) In vitro kinase assay of ROCK2^WT, ROCK2^T240A (non-phosphorylatable) and ROCK2^T240E (phosphomimetic). Error bars are the s.d. of three independent measurements. All three constructs show the same specific activity against MLC2.