Abstract. The three mammalian ras proteins associate specifically with the plasma membrane and this is essential for their biological activity. Two signals encoded within the extreme COOH terminus of the proteins specify this cellular localization; a CAAX box in combination with either a polybasic domain (p21K-~B) or a palmitoylation site (p21 m-~ and p21N-~). All members of the ras-like and rho-like subfamilies of the ras superfamily of small GTP-binding proteins also have CAAX boxes with potential second site sequences resembling either p21K-~B or P21N-~m-~. However it is not at all clear that they are each located at the plasma membrane, and in fact one of the ras-like proteins, rapl, has been localized to the Golgi (Beranger et al., 1991). None of the mammalian rho-like subfamily has yet been localized. Three forms (A, B, and C) of p21 ~h~ the prototype of this family are known; the COOH termini of p21 rh~ and p21 rh~ resemble p21K-'~B with a polybasic domain, whereas p21 rh~ resembles p21N-r~/m-r~ with two cysteine residues as potential palmitoylation sites. Despite this similarity to the p21 ~ proteins, rho proteins have been purified from both particulate and cytosolic fractions of a variety of tissues. In order to localize definitively the three rho proteins we have used an epitope tagging approach coupled to microinjection of living cells. We show that a small fraction of all three proteins is localized to the plasma membrane but the majority of p21 rh~ and p21 rh~ is cytosolic whereas p21 ~h~ is associated with early endosomes and a pre-lysosomal compartment. Along with the results obtained with chimeric molecules using heterologous proteins attached to rho COOH termini, this suggests that the p2P h~ proteins cycle on and off the plasma membrane and this may have important implications for their biological function.

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Endothelium of the cerebral blood vessels, which constitutes the blood-brain barrier, controls adhesion and trafficking of leukocytes into the brain. Investigating signaling pathways triggered by the engagement of adhesion molecules expressed on brain endothelial cells using two rat brain endothelial cell lines (RBE4 and GP8), we report in this paper that ICAM-1 cross-linking induces a sustained tyrosine phosphorylation of the phosphatidylinositol-phospholipase C (PLC)γ1, with a concomitant increase in both inositol phosphate production and intracellular calcium concentration. Our results suggest that PLC are responsible, via a calcium- and protein kinase C (PKC)-dependent pathway, for p60Src activation and tyrosine phosphorylation of the p60Src substrate, cortactin. PKCs are also required for tyrosine phosphorylation of the cytoskeleton-associated proteins, focal adhesion kinase and paxillin, but not for ICAM-1-coupled p130Cas phosphorylation. PKC’s activation is also necessary for stress fiber formation induced by ICAM-1 cross-linking. Finally, cell pretreatment with intracellular calcium chelator or PKC inhibitors significantly diminishes transmonolayer migration of activated T lymphocytes, without affecting their adhesion to brain endothelial cells. In summary, our data demonstrate that ICAM-1 cross-linking induces calcium signaling which, via PKCs, mediates phosphorylation of actin-associated proteins and cytoskeletal rearrangement in brain endothelial cell lines. Our results also indicate that these calcium-mediated intracellular events are essential for lymphocyte migration through the blood-brain barrier.

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Summary Lymphocytes emigrate from the circulation to target tissues through the microvascular endothelial cell (EC) barrier. During paracellular transmigration cell-cell junctions have been proposed to disengage and provide homophilic and heterophilic interaction surfaces in a zip-like process. However, it is not known whether EC modulate junction proteins during this process. Here we show that tyrosine phosphorylation of adherens junction vascular endothelial cadherin (VEC) is required for successful transendothelial lymphocyte migration. We found that adhesion of lymphocytes or activation of the endothelial adhesion-receptor ICAM-1 led to tyrosine phosphorylation of VEC. Substitution of tyrosine to phenylalanine in VEC at position 645, 731 or 733 produced EC which were significantly less permissive to lymphocyte migration. We also found that these same tyrosines were involved in ICAM-1-dependent changes of VEC phosphorylation. ICAM-1 activation enhanced transendothelial permeability suggesting that junction disassembly occurred. In agreement the expression of Y645F, Y731F or Y733F VEC predominantly affected lymphocyte transmigration in paracellular areas. Taken together these results demonstrate that adherens junction phosphorylation constitutes a molecular endpoint of lymphocyte-induced vascular EC signaling and may be exploited as a new target of anti-inflammatory therapies.