Abstract
Centrosome- and Golgi-localized protein kinase N-associated protein (CG-NAP), also known as AKAP450, is a cytosolic scaffolding protein involved in the targeted positioning of multiple signaling molecules, which are critical for cellular functioning. Here, we show that CG-NAP is predominantly expressed in human primary T-lymphocytes, localizes in close proximity (<0.2 μm) with centrosomal and Golgi structures and serves as a docking platform for Protein Kinase A (PKA). GapmeR-mediated knockdown of CG-NAP inhibits LFA-1-induced T-cell migration and impairs T-cell chemotaxis toward the chemokine SDF-1α. Depletion of CG-NAP dislocates PKARIIα, disrupts centrosomal and non-centrosomal microtubule nucleation, causes Golgi fragmentation, and impedes α-tubulin tyrosination and acetylation, which are important for microtubule dynamics and stability in migrating T-cells. Furthermore, we show that CG-NAP coordinates PKA-mediated phosphorylation of pericentrin and dynein in T-cells. Overall, our findings provide critical insights into the roles of CG-NAP in regulating cytoskeletal architecture and T-cell migration.
Highlights
The migration of T-lymphocytes, i.e., their homing to lymphoid organs, recruitment to inflamed tissue sites and mounting an adaptive immune response against infection, is a complex, but precisely regulated physiological process
Confocal microscopy shows that the majority of this adaptor protein is contained at the centrosomal region as distinct spots with some amount of the protein in the cytoplasm and on the membrane in both resting and LFA-1-stimulated peripheral blood lymphocyte (PBL) T-cells (Figure 1B). 3D-Structured Illumination (3D-SIM) imaging and subsequent volume-rendered reconstruction analysis reveal that about 12% of centrosomally located Centrosome- and Golgi-localized protein kinase N-associated protein (CG-NAP) protein spots are present in close proximity (
To determine whether CG-NAP is essential for T-cell migration, we knocked down the expression of this protein in HuT78 T-cells (>90% KD) using our recently reported GapmeR-mediated gene silencing technique [21] (Figure 2A)
Summary
The migration of T-lymphocytes, i.e., their homing to lymphoid organs, recruitment to inflamed tissue sites and mounting an adaptive immune response against infection, is a complex, but precisely regulated physiological process. This requires coordinated signal transduction pathways that culminate in dynamic reorganization of the cytoskeletal systems and active T-cell locomotion. Radial arrays of highly dynamic microtubule plus ends extend from the centrosome toward the cell periphery that ensures regulated signal transduction and rapid cell migration. It has recently been noted that microtubule nucleation can arise via centrosome-independent mechanisms from membranous organelles, contributing to the asymmetric microtubule networks in polarized cells [1]
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