The metalloprotease-dependent extracellular domain cleavage of the adhesion molecule CD44 is frequently observed in human tumors and is thought to promote metastasis. This cleavage is followed by ;-secretase-dependent release of CD44 intracellular domain (CD44-ICD), which exhibits nuclear signaling activity. Using a reversible Ret-dependent oncogenic conversion model and a restricted proteomic approach, we identified a positive correlation between the neoplastic transformation of Rat-1 cells and the expression of standard CD44. In these transformed cells, CD44 was found to undergo a sequential metalloprotease and ;-secretase cleavage, resulting in an increase in expression of CD44-ICD. We showed that this proteolytic fragment possesses a transforming activity. In support of this role, a significant and specific reduction in Ret-induced transformation of Rat-1 cells was observed following drug-mediated inhibition of ;-secretase. Taken together, these findings suggest that the shedding of CD44 may not only modulate metastasis but also affects earlier events in tumorigenesis through the release of CD44-ICD.

Dominant-activating mutations in the RET (rearranged during transfection) proto-oncogene, a receptor tyrosine kinase, are causally associated with the development of multiple endocrine neoplasia type 2A (MEN2A) syndrome. Such oncogenic RET mutations induce its ligand-independent constitutive activation, but whether it spreads identical signaling to ligand-induced signaling is uncertain. To address this question, we designed a cellular model in which RET can be activated either by its natural ligand, or alternatively, by controlled dimerization of the protein that mimics MEN2A dimerization. We have shown that controlled dimerization leaves proximal RET signaling intact but impacts substantially on the tuning of the distal AKT kinase activation (delayed and sustained). In marked contrast, distal activation of ERK remained unaffected. We further demonstrated that specific temporal adjustment of ligand-induced AKT activation is dependent upon a lipid-based cholesterol-sensitive environment, and this control step is bypassed by MEN2A RET mutants. Therefore, these studies revealed that MEN2A mutations propagate previously unappreciated subtle differences in signaling pathways and unravel a role for lipid rafts in the temporal regulation of AKT activation.The RET 4 proto-oncogene is located on chromosome 10q11.2 and encodes a receptor tyrosine kinase with four cadherin-related motifs and a cysteine-rich domain in the extracellular domain (1). It associates with ligand-specific co-receptors known as GFR␣s (GDNF, glial-cellline-derived neurotrophic factor, family receptors ␣), to form functional receptors for the GFL (GDNF family ligands). In the current view, homodimeric GFL binding induces a GFL 2 -GFR␣ 2 -RET 2 complex (2). RET dimerization leads to increased receptor kinase activity and autophosphorylation of cytoplasmic tyrosine residues, which serve as docking sites for Src homology 2 (SH2)-and phospho-tyrosine-binding domain-containing proteins, such as Shc or phospholipase C␥ (3). These proteins then recruit additional effector molecules, resulting in the assembly of signaling complexes and the activation of intracellular signaling pathways, including the Ras/extracellular-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)/AKT pathways. GFL-mediated signaling pathways are involved in the development and maintenance of a broad spectrum of neuronal subpopulations (1).Recently, membrane microdomains, or lipid rafts, have been shown to profoundly influence the functional impact of GDNF-stimulated RET downstream signaling (4, 5). Lipid rafts are suggested to be lateral microdomains in membranes of living cells, enriched in sphingolipids, cholesterol, and specific membrane proteins. They are characterized by higher order and by having a lower buoyant density than bulk plasma membranes (6, 7). Although uncertainties about the precise molecular nature of rafts remain (8 -11), compelling evidence indicates that lipid rafts can coalesce into larger and more stable structures where proteins can segregate to perform...

Engagement of the T-cell receptor (TCR)results IntroductionT-cell immune responses are coupled to activation of the T-cell receptor (TCR). Stimulation of this receptor results in the activation of signal transduction pathways, culminating in expression of cytokines and cellular proliferation. 1 Current models of antigen/ major histocompatibility complex (MHC)-induced T-cell activation are presented as ordered events with a sequential interaction of Src and ZAP-70/Syk protein tyrosine kinases (PTKs) with the TCR/CD3/ complex. Specifically, TCR engagement activates the Src family PTKs Lck/Fyn, which phosphorylate the tyrosines present in the immunoreceptor tyrosine activation motif (ITAM) [2][3][4] conserved in the CD3 and subunits of the TCR complex. 5,6 The ZAP-70/Syk PTKs then bind to the phosphorylated ITAMs via their respective SH2 domains, allowing their activation. 2,7,8 Once activated, ZAP-70/Syk kinases phosphorylate downstream, signaling intermediates such as Vav, Lat, and SLP-76 that are required for appropriate recruitment of downstream signaling cascades.ZAP-70 and Syk are structurally homologous; both proteins are composed of 2 tandemly arranged Src homology 2 (SH2) domains and a carboxy-terminal kinase domain. 7,9 Overall, these 2 kinases share more than 50% sequence identity with conserved tyrosine and serine phosphorylation sites. Although these 2 PTKs have some overlapping functions, 1 it has been hypothesized that in vivo, they are nonredundant because of their distinct expression profiles. ZAP-70 was initially reported to be expressed exclusively in thymocytes, T cells, and natural killer (NK) cells, whereas Syk was described as being expressed in a wide variety of hematopoietic cells but in only low levels in peripheral T cells. 10 Nevertheless, it has since been shown that Syk is expressed at high levels in human CD4 ϩ effector T cells and a subpopulation of TCR-stimulated ␣␤ T cells. 1,11 Moreover, the expression of Syk is not limited to hematopoietic cells, as it has been detected in vascular endothelial cells, 12 in epithelial cells, and in breast tissue. 13 With regard to ZAP-70, this PTK now has been shown to be expressed throughout normal B-cell development, at least in mice. 14 In humans, ZAP-70 has recently been detected in a subset of chronic lymphocytic leukemia (CLL) B cells, and importantly, its expression has been correlated with a poor prognosis. [15][16][17] The increased phosphorylation of Syk in ZAP-70-expressing CLL cells was the first indication that ZAP-70 may modulate the activity of Syk 18,19 rather than vice versa. Indeed, until these recent data were reported, Syk was assumed to be "superior" to ZAP-70, as its kinase activity is 100-fold higher than that of 20 it can be activated in an Lck-independent fashion, 21-25 and TCR-induced calcium flux is higher in the presence of Syk than 27 It has previously been shown that both Syk and ZAP-70 bind to Lck [28][29][30] and that in vitro, the 2 kinases bind to chain ITAMs with similar affinities. 31-33 Indeed, it has been hypo...