THE SCAFFOLDING PROTEIN CG-NAP/AKAP450 IS A CRITICAL INTEGRATING COMPONENT OF THE LFA-1 INDUCED SIGNALING COMPLEX IN MIGRATORY T CELLS.

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It has become clear that in many cells, the process of signal transudation depends on cytoskeletal scaffolding proteins that directly coordinate signaling. By targeting effector kinases, phosphatases and substrates at the same subcellular location, these scaffolding proteins organize multiple signaling pathways within cells (20, 21, 22, 23, 24, 25). Moreover, it has been  shown that AKAPs play a role in the maintenance of T cell homeostasis (37).

CG-NAP/AKAP450 is a scaffolding protein from the family of related A-kinase anchoring proteins (AKAPs), which was shown to associate with the centrosome and the Golgi apparatus in a number of mammalian cell lines (30). CG-NAP/AKAP450 structurally represents a giant molecule with four leucine zipper-like motifs, a binding domain that interacts with PKA through its regulatory subunit PKA II, and a docking site for Rho-activated protein kinase PKN. CG-NAP/AKAP450has binding sites for protein phosphatases PP1, PP2A and  also found to interact with an immature non-phosphorylated form of PKCe (33). The complex coiled-coil architecture of the molecule adds to the characteristic features of CG-NAP/AKAP450 likely to be involved in multiple associations with several proteins.

In this study, we show for the first time that the scaffolding protein CG-NAP/AKAP450 is expressed in peripheral blood lymphocytes PBTLs and in the T lymphoma cell line HUT-78. Expression of this molecular scaffold is localized to the region of the centrosome and the Golgi apparatus. LFA-1 crosslinking results in the development of signaling complex that contains cytoskeletal components along with the protein kinase C isoenzymes b and d. We demonstrate here that CG-NAP/AKAP450 represents an integral component of this complex. Specifically by linking together the centrosome, microtubules and two enzymes involved in cytoskeletal modification in locomotory T cells, CG-NAP/AKAP450 could potentially contribute to the orchestration of LFA-1-induced intracellular signaling cascades. The functional importance of this is further underscored by the finding that the association is greatly reduced when cells are maintained at low temperature conditions hence indicating an active metabolic requirement for this association.

The LFA-1 cytoskeletal interactions are coordinated during lymphocyte migration. Association of CG-NAP/AKAP450 with the LFA-1 signaling complex may be dependent on intact microtubule functioning. Disruption of microtubules caused partial dispersion of CG-NAP/AKAP450 localization (data not shown), implying that this association may be important in at least some of the signaling process involved in migration (38). We have previously demonstrated that LFA-1 activation also induces the secretion of chemokines involved in regulation of directional cell migration (39). Such secretion would be predicted to be dependent on the Golgi apparatus function and also on the maintenance of cell polarity. Hence, it is possible that the generation of this signaling complex brings together not only molecules involved in migration but also molecules involved in intracellular transport and secretion. In addition to the critical role of PKCb in T cell migration, we have also demonstrated that PKCb expression is crucial for transport and secretion of the cytokine interleukin-2, further emphasizing the connectivities between the signaling events involved in these cellular functions. Microtubules play an active role in both migration and secretion. The association of CG-NAP/AKAP450 with the g-tubulin ring complex at the centrosome (40) suggests the possibility that CG-NAP/AKAP450 may play a crucial role in microtubule-dependent functions in both processes. 

The functional significance of CG-NAP/AKAP450 for LFA-1 induced motility is further underscored by the results of the studies in cell transfectants. The fact that cells over expressing C-terminal domain of CG-NAP/AKAP450 failed to polarize and acquire a locomotory phenotype indicates that CG-NAP/AKAP450 is crucially involved in one of the MTOC dependent cell functions such as active motile  behavior. These data are in concert with the recent findings by Keryer et al (34), who demonstrated that overexpression of CG-NAP/AKAP450 in Hela cells impairs cytokinesis and increases ploidy implying a significant functional role of CG-NAP/AKAP450 in the integrity of the centrosome and related signaling pathways.

In addition, the results of  studies in the fruit fly demonstrate that when the Drosophila homolog of CG-NAP/AKAP-450 (Drosophila peri-centrin-like protein D-PLP) is mutated, mitosis is not severely perturbed. However, D-PLP is essential for microtubule-supported cilia/flagella function and d-plp-mutant flies are uncoordinated and have non-functional neurons (41). Furthermore, pericentrin which shares homology to CG-NAP/AKAP450 has recently been shown to scaffold PKC to the centrosome and to control major centrosomal functions including microtubule organization, spindle function and cytokinesis (42).

 The findings that intracellular distribution of CG-NAP/AKAP450 was typical of the cells triggered via ICAM-1, but not fibronectin indicate that the involvement of this scaffolding protein might play a key role for b2 integrin T cell motility but may not necessarily play a similar role in adhesion and motility induced via different integrin ligands, for example, the b1 integrin ligand fibronectin or corresponding antibodies. This is further supported by the data showing that the amount CG-NAP/AKAP450 associated with fibronectin ligands in migrating cells was significantly lower in comparison to that associated with LFA-1 in similar experimental conditions. 

The fact that LFA-1 and wild type CG-NAP/AKAP450 no longer co-localized in the cells overexpressing C-terminal GFP-tagged CG-NAP/AKAP450, strongly indicates that the formation and function of the motility-associated protein complex containing both LFA-1 and CG-NAP/AKAP450 was inhibited under these conditions.

Taken together, our findings provide first definitive evidence that the protein CG-NAP/AKAP450 is a key scaffolding component of the multi-molecular complex assembled in T cells upon LFA-cross-linking and is functionally indispensable for cell polarity and migration induced by this integrin.