Figure 8.

Functional role of PIMT binding to CAIX: (a) Effect of Pcmt1 transient silencing on CAIX-mediated extracellular acidification. The graph shows the differences between pH values (ΔpH) measured in culture medium at the beginning and after 48 h in hypoxia normalized to protein concentration. Results clearly showed that transient Pcmt1 silencing in C33a-CAIX cells (stable CAIX transfectants) resulted in the alkalinization of extracellular pHe when compared to scramble control (scr). Measurements were performed after 72 h post-transfection of siRNAs. The data are presented as the mean ± s.d., n = 6. Statistical significance was analyzed using the Student’s t-test and expressed as a p-value (* p < 0.05). (b) Efficacy of transient silencing was checked in parallel at the protein level using immunoblot analysis depicted on a representative image. (c) Activation of CAIX enzymatic function under hypoxic conditions. Posttranslational modification of the intracellular tail is necessary for full activation of CAIX enzymatic function. Phosphorylation of Thr443 was identified previously. In this work we found out a novel condition necessary for CAIX activation—interaction between the IC tail of CAIX and PIMT facilitated by terminal Ala459. Hypoxia can affect the activity of CAIX in several ways: it upregulates cAMP levels through ADCY6 and ADCY7, leading to higher levels of cAMP, PKA activation and Thr443 phosphorylation, and also enhances the expression of PIMT, allowing its interaction with the IC tail of CAIX.