Functional analysis of Gimap4 motifs. (A) Gimap4 protein was produced in an in vitro TS/TL system. A pulldown of the protein with CaM beads in the absence (–) or presence (+) of Ca2+ was used to determine the binding capacity of CaM to Gimap4. A functional mutant (IQ*), where the 3 arginine residues (R) within the IQ domain were substituted for 3 glutamine residues (Q), failed to bind CaM. Furthermore, the empty vector was used as a negative control (NC). (B) Phosphorylation level of Gimap4 in the presence or absence of ConA (2 mg/mL). (C) Equal amounts of cell lysates from T cells treated with 10 ng/mL PMA plus 1 ng/mL ionomycin in the absence or presence of the PKC inhibitor rottlerin (1 mM) for 20 minutes were used for 2D gel analysis, and immunoblots were performed using anti-Gimap4 polyclonal antibody. Phospho-Gimap4 species are identified by a shift in the isoelectric point. In vitro inhibition of PKC activity abrogates phosphorylation of Gimap4. (D) Gimap4 phosphorylation diminishes after 60 minutes of PMA/ionomycin stimulation.
