Crucial importance of PKC-β(I) in LFA-1–mediated locomotion of activated T cells

103

Paxillin is a focal adhesion-associated protein that functions as a multi-domain adapter protein, binding several structural and signaling molecules. ␣-Tubulin was identified as an interacting protein in a two-hybrid screen using the paxillin C-terminal LIM domain as a bait. In vitro binding assays with glutathione S-transferase-paxillin demonstrated an interaction of ␣-tubulin with the C terminus of paxillin. Another member of the tubulin family, ␥-tubulin, bound to both the N and the C terminus of paxillin. The interaction between paxillin and both ␣-and ␥-tubulin in vivo was confirmed by coimmunoprecipitation from human T lymphoblasts. Immunofluorescence studies revealed that, in adherent T cells, paxillin localized to sites of cell-matrix interaction as well as to a large perinuclear region. Confocal microscopy revealed that this region corresponds to the lymphocyte microtubule organizing center, where paxillin colocalizes with ␣-and ␥-tubulin. The localization of paxillin to this area was observed in cells in suspension as well as during adhesion to integrin ligands. These data constitute the first characterization of the interaction of paxillin with the microtubule cytoskeleton, and suggest that paxillin, in addition to its well established role at focal adhesions, could also be associated with the lymphocyte microtubule network.

Section: Discussionmentioning

confidence: 73%

Paxillin Localizes to the Lymphocyte Microtubule Organizing Center and Associates with the Microtubule Cytoskeleton

Herreros¹,

Rodrı́guez-Fernández²,

Brown³

et al. 2000

103

“…Activated PKC isoforms have been shown to bind microtubules in T cells, U923 cells, and Aplysia neurons, and the results suggested that they may play a role in regulating microtubule dynamics or stability (19,42,43). A role for PKC in orienting the centrosome has also been suggested in studies of Drosophila embryos and T lymphocytes (44,45).…”

Section: Discussionmentioning

confidence: 97%

“…Many of these changes result from alterations in cytoskeletal structure, and several lines of evidence implicate PKC in regulating microtubule function. First, a number of microtubule-associated proteins are substrates for PKC both in vitro and in vivo (13,14); second, phorbol esters cause microtubule reorganization (15,16); third, PKC ␤I has been shown to co-localize with microtubules and bind microtubule-associated proteins (15); and fourth, novel PKC isoforms ⑀ and ␦ have been reported to localize to the centrosome (17,18). Importantly, recent studies demonstrate that PKC ␤-deficient T cells failed to develop a polarized microtubule network, a defect that can be rescued by expressing PKC ␤I (19).…”

mentioning

confidence: 99%

Centrosomal Anchoring of Protein Kinase C βII by Pericentrin Controls Microtubule Organization, Spindle Function, and Cytokinesis

Chen

Purohit

Halilovic

et al. 2004

Location is a critical determinant in dictating the cellular function of protein kinase C (PKC). Scaffold proteins contribute to localization by poising PKC at specific intracellular sites. Using a yeast two-hybrid screen, we identified the centrosomal protein pericentrin as a scaffold that tethers PKC ␤II to centrosomes. Co-immunoprecipitation studies reveal that the native proteins interact in cells. Co-overexpression studies show that the interaction is mediated by the C1A domain of PKC and a segment of pericentrin within residues 494 -593. Immunofluorescence analysis reveals that endogenous PKC ␤II colocalizes with pericentrin at centrosomes. Disruption of this interaction by expression of the interacting region of pericentrin results in release of PKC from the centrosome, microtubule disorganization, and cytokinesis failure. Overexpression of this disrupting fragment has no effect in cells lacking PKC ␤II, indicating a specific regulatory role of this isozyme in centrosome function. These results reveal a novel role for PKC ␤II in cytokinesis and indicate that this function is mediated by an interaction with pericentrin at centrosomes.

“…PKC␤II has previously been shown to bind and be activated by cytoskeletal elements such as F-actin (62). Additionally, T-cell initiation of crawling locomotion via integrin receptors is dependent on the phosphorylation of microtubules by PKC␤I (63). Whatever mechanism PKC␤ regulates, it may serve as a specific target for inhibiting human monocyte chemotaxis in different disease processes while possibly not affecting neutrophil migration or monocyte migration to other chemokines.…”

Section: Discussionmentioning

confidence: 99%

Protein Kinase C β Is Required for Human Monocyte Chemotaxis to MCP-1

Carnevale

Cathcart

2003

Monocyte chemoattractant protein 1 (MCP-1) is important in attracting monocytes to sites of inflammation. Using predominantly pharmacological approaches, prior studies have indicated that serine/ threonine kinases are involved in the MCP-1-induced signaling pathways. We report here that there is substantial inhibition of MCP-1-stimulated chemotaxis of human monocytes treated with inhibitors selective for the subset of serine/threonine kinases, protein kinase C (PKC). Selective inhibitors of PKC such as GF109203X and Calphostin C both caused ϳ80% inhibition of chemotaxis. Because these pharmacological inhibitors do not specifically inhibit individual PKC isoforms, we chose to use antisense oligodeoxyribonucleotides (ODN) to specifically reduce PKC isoform expression, first by inhibiting expression of the conventional PKC family, and next by using specific antisense ODN for PKC␣ and PKC␤. Conventional PKC-antisense ODN treatment completely and significantly inhibited monocyte chemotaxis to MCP-1, whereas sense-control ODN caused no significant inhibition. PKC␤-antisense ODN caused 89.2% inhibition of chemotaxis at its highest dose. In contrast, PKC␤-sense ODN and PKC␣-antisense and -sense ODN were without effect. Further studies evaluating the calcium response that is triggered upon MCP-1 interaction with its receptor, CCR 2 , indicate that this response is not altered by antisense or sense ODN treatment, thus supporting our hypothesis that PKC␤ is critical for post-receptor signal transduction downstream of the immediate calcium signal. These data contribute to our developing understanding of the signal transduction pathways involved in the chemotactic response of human monocytes to MCP-1 and uniquely identify the requirement for the PKC␤ isoform in this important process.