Figure 2: ARHGAP36 interacts with PKAC via a pseudosubstrate domain.
(a) HEK293T cells were transfected with PKAC-YFP together with Flag-tagged wild-type ARHGAP36 (WT), 195–516 (ΔN), 1–194 (N) or Flag-Cherry control. Lysates were immunoprecipitated using a GFP antibody and immunoblotted using GFP or Flag antibodies. (b) Confocal live micrographs of MDCK cells expressing PKAC-YFP together with CFP-ΔN, CFP-N or CFP-N2 (118–194). Nuclear enrichment of ARHGAP36-N2, bottom left, may be facilitated by the arginine-rich sequence. Scale bars, 10 μm. (c) Immobilized peptide ‘spots’, overlapping 25-mer peptides each shifted along by 5 aa in the entire ARHGAP36 sequence, were probed for interaction with GST-PKAC and immunoblotted using a GST antibody. The sequence of the spot with strongest interaction is shown. (d) Alanine and aspartate scans of the spot indicated in c were treated the same as in c. Asterisk indicates wild-type sequence spot. (e) Alignment of human ARHGAP36 with the human isoforms of PRKAR and PKI revealing its pseudosubstrate motif RRxAY. (f) Confocal live micrographs of MDCK cells coexpressing CFP-ARHGAP36-RRV and PKAC-YFP. Scale bars, 10 μm. (g) Average Venus-YFP fluorescence lifetimes of MDCK cells expressing PKAC-YFP alone or together with mCherry-ARHGAP36, -ΔN or -RRV, or PKAC-EEE-YFP (E127A/E170A/E230A) together with mCherry-ARHGAP36 (n≥4 each). Error bars denote mean±s.d. ***P<0.001, *P<0.05; NS, not significant; Student’s T-test versus PKAC-YFP donor alone. (h) ITC measurements were performed in the presence of 2 mM ATP analogue adenylyl-imidodiphosphate (AMPPNP). A concentration of 30 μM His-PKAC solution (cell) was titrated with 507 μM PKI or 351 μM 36i (syringe) until saturation was reached using 8 or 6 μl injections, respectively (the first injection was always carried out with half the volume and omitted from data analysis). 36i and PKI bound to PKAC●AMPPNP with almost identical affinities. The measured values are in a similar range as previously reported for the PKAC–PKI interaction in the presence of other ATP analogues or ADP71.
