The evolutionarily conserved Ras/mitogen-activated protein kinase (MAPK) cascade is an integral part of the processes of cell division, differentiation, movement and death. Signals received at the cell surface are relayed into the nucleus, where MAPK phosphorylates and thereby modulates the activities of a subset of transcription factors. Here we report the cloning and characterization of a new component of this signal transduction pathway called Mae (for modulator of the activity of Ets). Mae is a signalling intermediate that directly links the MAPK signalling pathway to its downstream transcription factor targets. Phosphorylation by MAPK of the critical serine residue (Ser 127) of the Drosophila transcription factor Yan depends on Mae, and is mediated by the binding of Yan to Mae through their Pointed domains. This phosphorylation is both necessary and sufficient to abrogate transcriptional repression by Yan. Mae also regulates the activity of the transcriptional activator Pointed-P2 by a similar mechanism. Mae is essential for the normal development and viability of Drosophila, and is required in vivo for normal signalling of the epidermal growth factor receptor. Our study indicates that MAPK signalling specificity may depend on proteins that couple specific substrates to the kinase.

Human protein S (PS), a cofactor of anticoagulant-activated protein C (APC), is a modular protein containing 4 epidermal growth factor (EGF)-like domains. EGF1 appears to mediate PS interaction with APC, but the roles of EGFs 2, 3, and 4 are less clear. We synthesized PS variants lacking single EGF domains (EGF2, 3, or 4) and assessed their APC cofactor activity in a factor Va inactivation assay. The variant lacking EGF2 (variant 134) showed the most dramatic loss of activity (ϳ10% of recombinant wild-type PS activity). Replacement of EGF2 by an additional EGF3 (variant 1334) resulted in a comparable loss of activity, suggesting that the loss of a specific rather than "spacer" function of EGF2 was responsible. We confirmed that the variant 134 had a functional ␥-carboxyglutamic acid (Gla) domain and that EGF1 was correctly folded. This is the first clear evidence that EGF2 is required for the expression of PS activity. (Blood.

Most in vitro investigations of endothelial cell function have been based on the behaviour of human umbilical vein endothelial cells (HUVEC) in primary tissue culture. However, it is becoming apparent that there is a marked degree of heterogeneity among endothelial cells derived from different vascular beds. We have studied primary HUVEC from 45 umbilical cords. Contrary to previously published reports, we were unable to detect ABO antigens on the surface of cultured HUVEC from group A or B cords. This was not due to an absence of precursor H substance which was uniformly expressed on HUVEC in primary tissue culture. Further investigation revealed an absence of A enzyme activity in A type HUVEC and a level of mRNA only just detectable by RT-PCR. The absence of functional A enzyme activity was confirmed by demonstrating the absence of A substance on von Willebrand factor secreted by A type cells. HUVEC appear to be the only vascular endothelial cells that do not express ABO blood group antigens. We speculate that this may help protect the cord from maternal antibodies during gestation. The lack of ABO blood group antigens on HUVEC may significantly affect their surface function, and therefore care should be taken when extrapolating conclusions from results obtained with these cells (the most widely used human endothelial cell model in vitro) to the properties of adult endothelial cells.

We have identified 2 PROS1 missense mutations in the exon that encodes the vitamin K-dependent Gla domain of protein S (Gly11Asp and Thr37Met) in kindred with phenotypic protein S deficiency and thrombosis. In studies using recombinant proteins, substitution of Gly11Asp did not affect production of protein S but resulted in 15.2-fold reduced protein S activity in a factor Va inactivation assay. Substitution of Thr37Met reduced expression by 33.2% (P < .001) and activity by 3.6-fold. The Gly11Asp variant had 5.4-fold reduced affinity for anionic phospholipid vesicles (P < .0001) and decreased affinity for an antibody specific for the Ca 2؉ -dependent conformation of the protein S Gla domain (HPS21). Examination of a molecular model suggested that this could be due to repositioning of Gla29. In contrast, the Thr37Met variant had only a modest 1.5-fold (P < .001), reduced affinities for phospholipid and HPS21. This mutation seems to disrupt the aromatic stack region. The proposita was a compound heterozygote with free protein S antigen levels just below the lower limit of the normal range, and this is now attributed to the partial expression defect of the Thr37Met mutation. The activity levels were strongly reduced to 15% of normal, probably reflecting the functional deficit of both protein S variants. Her son (who was heterozygous only for Thr37Met) had borderline levels of protein S antigen and activity, reflecting the partial secretion and functional defect associated with this mutation. This first characterization of natural protein S Gla-domain variants highlights the importance of the high affinity protein S-phospholipid interaction for its anticoagulant role. IntroductionProtein S is a plasma glycoprotein that plays an important role in the protein C anticoagulant pathway by acting as a cofactor to activated protein C (APC) in the specific proteolytic inactivation of factors Va and VIIIa, reviewed in Simmonds and Lane. 1 In healthy individuals, ϳ60% of circulating protein S is found in complex with C4b binding protein, and only free protein S has APC cofactor activity. The physiological requirement for protein S is clearly demonstrated by the clinical manifestations of purpura fulminans in infants who lack detectable protein S at birth. 2 Heterozygous deficiency of protein S is found in 1% to 2% of consecutive patients with deep vein thrombosis 3 and has been classified into 3 subtypes. Type I (total and free protein S below the lower limit of a normal range) and type III (normal total protein S, but reduced free protein S levels) deficiencies are now commonly considered to be quantitative. 4 Type II deficiency is qualitative and is characterized by reduced activity in a specific functional assay.Mature protein S has a modular structure and consists of a vitamin K-dependent Gla domain that includes an aromatic stack region (residues 1 to 46), a region sensitive to cleavage by thrombin (residues 47 to 75), 4 domains homologous to epidermal growth factor (EGF-like domains, residues 76 to 242), and a region ho...

Tumor-associated macrophages (TAMs) are a major supportive component within neoplasms. Mechanisms of macrophage (MΦ) attraction and differentiation to a tumor-promoting phenotype, which is characterized by pronounced interleukin (IL)-10 production, are under investigation. We report that supernatants of dying cancer cells induced substantial IL-10 release from primary human MΦs, dependent on signaling through tyrosine kinase receptor A (TRKA or neurotrophic tyrosine kinase receptor type 1 (NTRK1)). Mechanistically, sphingosine-1-phosphate (S1P) release from apoptotic cancer cells triggered src-dependent shuttling of cytosolic TRKA to the plasma membrane via S1P receptor signaling. Plasma membrane-associated TRKA, which was activated by constitutively autocrine secreted nerve growth factor, used phosphatidylinositol 3-kinase (PI3K)/AKT and p38 mitogen-activated protein kinase (MAPK) signaling to induce IL-10. Interestingly, TRKA-dependent signaling was required for cytokine production by TAMs isolated from primary murine breast cancer tissue. Besides IL-10, this pathway initiated secretion of IL-6, tumor necrosis factor-α (TNF-α) and monocyte chemotactic protein-1 (MCP-1), indicating relevance in cancer-associated inflammation. Our findings highlight a fine-tuned regulatory system including S1P-dependent TRKA trafficking for executing TAM-like cell function in vitro as well as in vivo.