Fig 1: The C-terminal domains of HsFhod1 and HsFhod3 are methylated by HsPRMT7 in vitro.A, schematic diagram of HsFhod1 and HsFhod3 and constructs used in this paper. Conserved domains are indicated. Light green boxes in Fhod3 are inserts that distinguish isozyme 4 from isozyme 1. The dark blue box (RTRSR) contains a sequence similar to the PRMT7 recognition sequence (RKRSR), which is in the red box. Red asterisk (∗) denotes a serine that is phosphorylated by ROCK1/2. B, sequence alignment of HsFhod1 and HsFhod3 (isozymes 1 and 4) C-terminal residues including the core DAD domain (bold) and the PRMT7 recognition motif (red box). C, 7 μg of MBP-Fhod1-tail or 7 μg of Fhod3-CT, 5 μg of GST-HsPRMT7, and 0.7 μM [3H]AdoMet were combined and allowed to incubate for 20 h at 4 °C in a reaction buffer containing 1 mM DTT, 50 mM K-HEPES, with a final pH of 8.5 in a final volume of 30 μl. Samples were separated by SDS-PAGE electrophoresis and exposed to autoradiography film for 5 days. Single asterisk (∗) denotes the polypeptide molecular weight of MBP-Fhod1-tail; double asterisk (∗∗) denotes the polypeptide molecular weight of Fhod3-CT.
Fig 2: ROCK1 enzymatic activity is minimally affected by the presence of methylarginine residues in a peptide containing serine 1589 and serine 1595. 130 ng of ROCK1, 1 mM ATP and 10 μM Fhod3 (1581-1595) peptides were incubated in a water bath at 30 °C for the specified time in a final volume of 30 μl. Reactions were terminated with TFA and loaded into a vial for mass spectrometry analysis. 10 μl of the sample was injected into an Agilent QTOF 6545 with a run time of 12 min. A and B, Fhod3 WT, C and D, Fhod3 R1588Me1, E and F, Fhod3 R1590Me1, G and H, Fhod3 R1588Me1/R1590Me1. Change in the total amount of unmodified or phosphorylated peptide in reaction mixtures from charge states [M+2H] (black line), [M+3H] (green line), [M+4H] (blue line), and [M+5H] (red line) with time.
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