REGULATION OF THE ASSOCIATION OF CDC42 INTERACTING PROTEIN 4 (CIP4) WITH MICROTUBULES DURING THE ESTABLISHMENT OF THE IMMUNE SYNAPSE IN NATURAL KILLER CELLS: INVOLVEMENT OF PKA

HUHN V; PARIANI A; RIVABELLA MAKNIS T; FAVRE C; LAROCCA MC

Congreso; Reunión Anual de la Sociedad Argentina de Fisiología; 2023

Introduction: Natural killer (NK) cell cytotoxicity requires extensive actin and microtubule (MT) remodeling at the NK-target cell immune synapse. CIP4 is a CDC42 effector that scaffolds proteins involved in actin remodeling, which has a prominent role in NK-immune synapse maturation. CIP4 also contains an amino-terminal domain that allows CIP4 interaction with MT, and is phosphorylated by PKA in CIP4T225. Our previous results indicate that YTS-NK cell activation leads to decreased CIP4 association with MT. Objectives: We aimed to assess the relevance of our previous findings in ex vivo NK (eNK) cells and to analyze the involvement of PKA in the regulatory mechanism. Methods: eNK cells from volunteer blood donors were purified by negative selection and cultured in IL-2-supplemented medium. To evaluate the role of PKA, YTS-NK cells were cultivated in the presence of PKA activator forskolin (Fk, 10 µM) or PKA inhibitor H89 (1 µM). Both, eNK and YTS-NK cells, were exposed to erythroleukemia KT-86 cells for 30 min at 2:1 or 6:1 ratios for immunofluorescence (IF) or Western blot (WB) studies. CIP4 interaction with MT was assessed by IF confocal microscopy. An antibody that specifically recognizes residues phosphorylated by PKA was used to assess PKA activity in NK-cells exposed to target cells (activated, Ac) and in isolated NK cells (resting, R) by WB. Results are expressed as media ± SE. Results: IF studies showed that the fraction of MT associated CIP4 was reduced in Ac-eNK (MT-CIP4 (%), R: 17 ± 2; Ac: 13 ± 3; n=3; p