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The regulation of PTEN intrinsic biochemical properties has not been fully elucidated. In this report, we investigated the role of the PTEN carboxyl-terminal tail domain in regulating its membrane targeting and catalytic functions. Characterization of a panel of PTEN phosphorylation site mutants revealed that mutating Ser-385 to alanine (S385A) promoted membrane localization in vivo and phosphatase activity in vitro. Furthermore, S385A mutation was associated with a substantial reduction in the phosphorylation of the Ser-380/Thr-382/Thr-383 cluster. Therefore, Ser-385 could prime additional dephosphorylation events to regulate PTEN catalytic activity. Moreover, substituting Ser-380/Thr-382/Thr-383 to phosphomimic residues reversed the phosphatase activity of the S385A mutation. Next, we further defined the underlying mechanisms responsible for the COOH-terminal tail region in modulating PTEN biological activity. We have identified an interaction between the 71-amino acid carboxyl-terminal tail region and the CBRIII motif of the C2 domain, which has been implicated in membrane binding. In addition, a synthetic phosphomimic peptide encompassing the phosphorylation site cluster between amino acids 368 and 390 within the tail region mediated the suppression of PTEN catalytic activity in vitro. This same peptide when expressed in cultured cells also impeded PTEN membrane localization and enhanced phospho-Akt levels. Thus, our data suggest that the COOH-terminal tail can act as an autoinhibitory domain to control both PTEN membrane recruitment and phosphatase activity.

Homologue of Slimb (HOS) is the substrate-recognizing component of the SCF(HOS)-Roc1 E3 ubiquitin protein ligase. This ligase mediates ubiquitination of the inhibitor of NF-kappaB transcription factor (IkappaB). We have found that HOS is highly expressed in a number of human cancer cell lines. The rates of the HOS gene transcription as well as HOS mRNA and protein levels were up-regulated in cells treated with mitogens or transfected with the inducers of mitogen-activated protein kinase pathway. Conversely, mitogen withdrawal strikingly reduced HOS levels during differentiation of mouse myoblasts. Activators of mitogen-activated protein kinase accelerated IkappaBalpha degradation and increased NF-kappaB transcriptional activity. Inhibition of HOS function via expression of dominant negative HOS (HOS(DeltaF)) initiated mouse myoblast differentiation and prevented Ras-mediated acceleration of IkappaBalpha degradation as well as NF-kappaB trans-activation and transformation of NIH3T3 cells. These data link the induction of HOS in proliferating cells with mitogen-signaling-dependent inhibition of cell differentiation and promotion of cell transformation.

We studied the regulation of three closely related members of Ras family G proteins, R-Ras, TC21 (also known as R-Ras2), and M-Ras (R-Ras3). Guanine nucleotide exchange of R-Ras and TC21 was promoted by RasGRF, C3G, CalDAG-GEFI, CalDAG-GEFII (RasGRP), and CalDAG-GEFIII both in 293T cells and in vitro. By contrast, guanine nucleotide exchange of M-Ras was promoted by the guanine nucleotide exchange factors (GEFs) for the classical Ras (Ha-, K-, and N-), including mSos, RasGRF, CalDAG-GEFII, and CalDAG-GEFIII. GTPase-activating proteins (GAPs) for Ras, Gap1(m), p120 GAP, and NF-1 stimulated all of the R-Ras, TC21, and M-Ras proteins, whereas R-Ras GAP stimulated R-Ras and TC21 but not M-Ras. We did not find any remarkable difference in the subcellular localization of R-Ras, TC21, or M-Ras when these were expressed with a green fluorescent protein tag in 293T cells and MDCK cells. In conclusion, TC21 and R-Ras were regulated by the same GEFs and GAPs, whereas M-Ras was regulated as the classical Ras.

Transforming growth factor (TGF)-beta1, a pleiotropic cytokine involved in regulating growth and differentiation, can exert both pro-apoptotic and anti-apoptotic effects depending on the cell type or circumstances. We observed that TGF-beta1 blocked apoptosis resulting from serum withdrawal in A549 human lung carcinoma cells. This was associated with suppression of JNK activation that occurs concomitant with the onset of apoptosis in the absence of TGF-beta1, suggesting that JNK plays an active role in the death process and that TGF-beta1 exerts its protective influence by altering JNK activity. Overexpression of a dominant negative mutant form of SEK1, an upstream activator of JNK, likewise suppressed JNK activation and inhibited apoptosis. Investigation of early events following TGF-beta1 treatment revealed an early induction and phosphorylation of c-Jun that was absent in cells subjected to serum withdrawal alone. That TGF-beta1-induced expression of c-Jun is important for survival was supported by the finding that overexpression of non-phosphosphorylatable dominant negative mutant c-Jun, c-Jun(S73A), attenuated the protective influence of TGF-beta1. Our findings suggest that JNK activation is a late but essential event in serum deprivation-induced apoptosis in A549 cells. TGF-beta1 prevents apoptosis, in part, through the early induction and phosphorylation of c-Jun, which in turn results in attenuated JNK activation.

Hepatocyte growth factor/scatter factor (HGF/SF) stimulates cell proliferation, motility, and morphogenesis by activation of its receptor, the c-Met tyrosine kinase. HGF/SF is structurally related to plasminogen, including an amino-terminal hairpin loop, four kringle domains, and a serine protease-like region. A truncated HGF/SF isoform, designated HGF/NK2, which extends through the second kringle domain and behaves as a competitive HGF/SF antagonist, was previously shown to be encoded by an alternative HGF/SF transcript. In this study, we describe a second naturally occurring HGF/SF variant, HGF/NK1, consisting of the HGF/SF amino-terminal sequence and first kringle domain. This product is encoded by a 2-kilobase alternative transcript containing intronic sequence that was contiguous with exon K1b. Analysis of baculovirus-expressed HGF/NK1 revealed that this isoform possesses the heparin binding properties of HGF/SF and modest mitogenic and scattering activity relative to HGF/SF. However, at a 40-fold molar excess, HGF/NK1 inhibited HGF/SF-dependent DNA synthesis. HGF/NK1 stimulated tyrosine phosphorylation of Met, and covalent affinity cross-linking demonstrated a direct HGF/NK1-receptor interaction. These findings establish that the HGF/SF gene encodes multiple alternative products, which include not only a mitogenic agonist (HGF/SF) and a pure antagonist (HGF/NK2) but also a molecule with partial agonist/antagonist properties.

A cDNA predicted to encode a transmembrane tyrosine kinase receptor with sequence features characteristic of known fibroblast growth factor (FGF) receptors was isolated from an expression library constructed from the human mammary epithelial cell line B5/589. This cDNA, designated cl44, encodes a product of 803 amino acid residues and was readily distinguishable from known FGF receptors. During the course of our studies, Partanen et al. (Partanen, J., Makela, T. P., Eerola, E., Korhonen, J., Hirvonen, H., Claesson, W. L., and Alitalo, K. (1991) EMBO J. 10, 1347-1354) isolated a new FGF receptor, designated FGFR4, from the human leukemia cell line, K562. Its amino acid sequence is identical to that of cl44 with the exception of 1 residue. The 5'-untranslated sequences of the two cDNAs diverged far upstream of the initiation codon. A myoblast line, L6E9, which lacks FGF receptors, was utilized to express high levels of FGFR4. We found, in contrast to Partenen et al., who reported only binding of acidic FGF, that FGFR4 bound both acidic and basic FGF with dissociation constants of 10-15 and 120 pM, respectively. No detectable binding of keratinocyte growth factor was observed. In studies aimed to determine whether FGF receptors contribute to the development of human tumors, we screened RNAs prepared from cell lines derived from a variety of solid tumors. High levels of the cl44 transcript were detected in 8 of 14 and 6 of 9 human mammary and kidney carcinomas, respectively, but only infrequently in other types of tumors. In contrast, FGFR1 was found to be frequently expressed in kidney, but not in breast tumor cells, suggesting a possible role for FGFR4 in human mammary cancer.