An important negative control mechanism in the signaling of epidermal growth factor (EGF) is the endocytosis and subsequent degradation of activated EGF receptors. Eps15 and its related partner Eps15R play a key role in clathrin-mediated endocytosis of transmembrane receptors. Upon EGF stimulation of the cell, Eps15 becomes both phosphorylated on tyrosine residues and monoubiquitinated. Although tyrosine phosphorylation of Eps15 has been implicated in EGF receptor internalization, the function of Eps15 ubiquitination is not known. Using a mutational approach, we have found that the second ubiquitin-interacting motif (UIM) of Eps15 and Eps15R is essential for their ubiquitination. This UIM partially overlaps with the recently characterized nuclear export signal in Eps15. We show that these two overlapping motifs have different structural requirements with respect to nuclear export signal versus ubiquitination signal activity. Our data demonstrate that the UIM does not contain the ubiquitin acceptor site but functions as a recruitment site for the ubiquitination machinery leading to the monoubiquitination of both Eps15 and Eps15R.

Tracheal epithelial cells and skin fibroblasts from different cystic fibrosis (CF) patients bearing the deltaF508 mutation of cystic fibrosis transmembrane conductance regulator (CFTR) released more arachidonic acid in response to bradykinin than do other CF and normal cells. Immortalized tracheal epithelial cell lines were used as models to study the mechanisms of this dysregulation. An 85 kD cytosolic phospholipase A2 (cPLA2) was found in these cells and bradykinin increased its binding to membranes of deltaF508 cells (CFT-2) but not to those of a double heterozygous CF cells (CFT-1), or of control cells (NT-1). The expression of G alpha(q)/11 protein was also increased in deltaF508 cells, with increased stimulation of phosphatidylinositol diphosphate-specific phospholipase C (PLC) by bradykinin, and an early, transient activation of mitogen-activated protein (MAP) kinase. As the binding of cPLA2 to membranes is Ca2+-dependent, the increased coupling to PLC could cause the hypersensitivity to bradykinin. Comparison of the effects of bradykinin to those observed with thapsigargin, an inhibitor of calcium reuptake, suggests that the increase of intracellular calcium is not the only mechanism involved in arachidonic acid release by bradykinin in deltaF508 cells. The lack of effect of calcium ionophore A23187 or TPA on arachidonic acid release from any of the cell lines suggested that activation needs a PKC-independent cPLA2 phosphorylation step, perhaps via MAP kinase activation. The binding of cPLA2 to membranes after bradykinin stimulation still occurred in CFT2 cells (deltaF508) homogenized in EDTA, suggesting that a membrane component plus increased intracellular calcium influenced cPLA2 anchoring to membranes. The defective processing of deltaF508 CFTR seems to increase cPLA2 stimulation by bradykinin, since the bradykinin-stimulated release of arachidonic acid is reversed by growing cells at 28 degrees C for 48 h. The deltaF508 mutation of CFTR appears to increase the stimulation of cPLA2 by Gq-mediated receptors in a PKC-independent and MAP kinase-dependent manner. Hence normal CFTR, or normally processed deltaF508 CFTR, inhibit cPLA2 stimulation. The greater reactivity of deltaF508 CFTR cells to inflammatory mediators might be part of the increased sensitivity of CF patients to lung inflammation.

No abstract

The 85 kDa cytosolic phospholipase A 2 (cPLA 2 ) plays a key role in liberating arachidonic acid from the sn-2 position of membrane phospholipids. When activated by extracellular stimuli, cPLA 2 undergoes calciumdependent translocation from cytosol to membrane sites which are still a matter of debate. In order to evaluate the effect of plasma membrane association on cPLA 2 activation, we constructed chimeras of cPLA 2 constitutively targeted to the plasma membrane by the N-terminal targeting sequence of the protein tyrosine kinase Lck (Lck-cPLA 2 ) or the C-terminal targeting signal of K-Ras4B (cPLA 2 -Ras). Constitutive expression of these chimeras in Chinese hamster ovary cells overproducing the a 2B adrenergic receptor (CHO-2B cells) did not affect the basal release of [ 3 H]arachidonic acid, indicating that constitutive association of cPLA 2 with cellular membranes did not ensure the hydrolysis of membrane phospholipids. However, Lck-cPLA 2 increased [ 3 H]arachidonic acid release in response to receptor stimulation and to increased intracellular calcium, whereas cPLA 2 -Ras inhibited it, compared with parental CHO-2B cells and CHO-2B cells producing comparable amounts of recombinant wild-type cPLA 2 . The lack of stimulation of cPLA 2 -Ras was not due to a decreased enzymatic activity as measured using an exogenous substrate, or to a decreased phosphorylation of the protein. These results show that the plasma membrane is a suitable site for cPLA2 activation when orientated correctly.