DIFFERENTIAL REQUIREMENT OF PROTEIN-TYROSINE KINASES AND PROTEIN-KINASE-C IN THE REGULATION OF T-CELL LOCOMOTION IN 3-DIMENSIONAL COLLAGEN MATRICES

Locomotion of T lymphocytes within three-dimensional collagen matrices is regulated via different signaling states of the cells. Purified hu man CD4(+) and CD8(+) T cells developed a spontaneously locomoting sub population of about 25% of the whole population immediately after inco rporation into a three-dimensional collagen matrix analyzed by time-la pse videomicroscopy. This spontaneous locomotion was accompanied by en hanced tyrosine phosphorylation of the focal adhesion kinase (FAK). In hibition of protein tyrosine kinase (PTK) activity using genistein sig nificantly reduced the spontaneous locomotory activity. This reduction was overcome by subsequent activation of protein kinase C (PKC) using PMA, which led to a persistent increase of locomotory activity to mor e than 60% of the cells. Thus, the PKC-driven type of locomotion was i ndependent of PTK activity, whereas spontaneous locomotion was not alt ered by inhibition of PKC activity using calphostin C or inhibition of the serine/threonine phosphatases pp1 and pp2A using okadaic acid. We presume that PTK activity, especially tyrosine phosphorylation of FAK , is decisively involved in the regulation of spontaneous T lymphocyte locomotion, which is independent of PKC activity. In contrast, PKC-dr iven locomotion is independent of tyrosine phosphorylation events, ind icating that T lymphocyte locomotion is regulated by more than one sig nal transduction pathway. Furthermore, confocal microscopy analysis of phosphotyrosine residues, FAK, and PKC revealed an exclusive cellular distribution of these components, suggesting a regulation of T lympho cyte locomotion different from migration models developed for other ce ll types, which refer to a colocalization of FAK and PKC in focal adhe sions.