We have used a c-Src-GFP fusion protein to address the spatial control of Src activation and the nature of Src-associated intracellular structures during stimulus-induced transit to the membrane. Src is activated during transit, particularly in RhoB-containing cytoplasmic endosomes associated with the perinuclear recycling compartment. Knocking out RhoB or expressing a dominant-interfering Rab11 mutant suppresses both catalytic activation of Src and translocation of active kinase to peripheral membrane structures. In addition, the Src- and RhoB-containing endosomes harbor proteins involved in actin polymerization and filament assembly, for example Scar1, and newly polymerized actin can associate with these endosomes in a Src-dependent manner. This implies that Src may regulate an endosome-associated actin nucleation activity. In keeping with this, Src controls the actin dependence of RhoB endosome movement toward the plasma membrane. This work identifies RhoB as a component of "outside-in" signaling pathways that coordinate Src activation with translocation to transmembrane receptors.

hnRNP K and hnRNP E1/E2 regulate human papilloma virus type 16 (HPV-16) L2 capsid protein mRNA and reticulocyte 15-lipoxygenase (LOX) mRNA expression in the course of cellular differentiation. The expression of the virus capsid protein L2 is restricted to terminally differentiated epithelial cells in the superficial layers of the squamous epithelium by repression of L2 mRNA translation in the deeper layers (13). The underlying inhibitory mechanism employs hnRNPs K and E1/E2 interacting with a specific cis-acting element in the 3Ј end of L2 mRNA (5). LOX is a key enzyme in erythroid cell differentiation. It can attack phospholipids of the mitochondrial membranes and participates in their breakdown in late reticulocytes (20,24). LOX expression is temporally restricted by translational silencing of LOX mRNA in erythroid precursor cells in the bone marrow and in early reticulocytes (12). The differentiation control element (DICE) in the 3Ј untranslated region (UTR) of LOX mRNA binds the KH domain proteins hnRNP K and hnRNP E1 to form translationally silenced mRNPs in immature erythroid cells (15,16). The hnRNP K/E1-DICE complex blocks 80S ribosome assembly by inhibition of 60S ribosomal subunit joining (15). This erythroid silencing mechanism can be recapitulated in vitro and in HeLa cells transfected with DICE-regulated reporter mRNAs and hnRNP K alone or together with hnRNP E1 (16). Like LOX mRNA silencing, the HPV-16 L2 mRNA mechanism has been shown to operate in HeLa cells as well (5).The C-terminal part of hnRNP K contains proline-rich domains, which enable hnRNP K to interact with the SH3 domains of members of the Src kinase family (3) such as c-Src itself (23,25,27), Fyn, and Lyn (27). c-Src and Lck, a further member of the Src kinase family, have been shown to be able to phosphorylate hnRNP K in vitro and to affect its binding to RNA (19). The functional significance of hnRNP K tyrosine phosphorylation by members of the Src family of kinases is as yet unknown.Here we show that hnRNP K specifically binds and activates c-Src. c-Src mediates tyrosine phosphorylation of hnRNP K and inhibition of its DICE binding activity. Moreover, we demonstrate that c-Src kinase specifically regulates hnRNP K function as a translational silencer in vivo. Our results identify a specific role of c-Src in posttranscriptional regulation via hnRNP K, and suggest a mechanism for how the differentiation-dependent translation of cellular and viral RNA could be activated in maturing cells. MATERIALS AND METHODSPlasmids. For luciferase (LUC) indicator constructs, the LUC cDNA from pGEM-LUC (Promega) was inserted into pSG5 (16). LUC-DICE and LUC-NR were made by insertion of the DICE or nonregulatory (NR) sequences of the LOX mRNA 3Ј UTR into the EcoRV site of pSG5 before insertion of the LUC open reading frame (18). pSG5-His-hnRNP K was generated using pSG5-hnRNP K (16) by insertion of an oligonucleotide coding for 10 histidine residues between the SmaI and XhoI sites, N-terminal to hnRNP K. The tyrosine-tophenylalanine mutants pSG5-His-...

Recently, we demonstrated that the expression levels of the translationally controlled tumor protein (TCTP) were strongly down-regulated at the mRNA and protein levels during tumor reversion͞suppression and by the activation of p53 and Siah-1. To better characterize the function of TCTP, a yeast two-hybrid hunt was performed. Subsequent analysis identified the translation elongation factor, eEF1A, and its guanine nucleotide exchange factor, eEF1B␤, as TCTP-interacting partners. In vitro and in vivo studies confirmed that TCTP bound specifically eEF1B␤ and eEF1A. Additionally, MS analysis also identified eEF1A as a TCTP interactor. Because eEF1A is a GTPase, we investigated the role of TCTP on the nucleotide exchange reaction of eEF1A. Our results show that TCTP preferentially stabilized the GDP form of eEF1A, and, furthermore, impaired the GDP exchange reaction promoted by eEF1B␤. These data suggest that TCTP has guanine nucleotide dissociation inhibitor activity, and, moreover, implicate TCTP in the elongation step of protein synthesis.

The p53 tumor suppressor protein plays a crucial role in tumorigenesis by controlling cell-cycle progression and apoptosis. We have previously described a transcript designated tumor suppressor activated pathway-6 (TSAP6) that is up-regulated in the p53-inducible cell line, LTR6. Cloning of the murine and human fulllength TSAP6 cDNA revealed that it encodes a 488-aa protein with five to six transmembrane domains. This gene is the murine and human homologue of the recently published rat pHyde. Antibodies raised against murine and human TSAP6 recognize a 50-to 55-kDa band induced by p53. Analysis of the TSAP6 promoter identified a functional p53-responsive element. Functional studies demonstrated that TSAP6 antisense cDNA diminished levels of the 50-to 55-kDa protein and decreased significantly the levels of p53-induced apoptosis. Furthermore, TSAP6 small interfering RNA inhibited apoptosis in TSAP6-overexpressing cells. Yeast two-hybrid analysis followed by GST͞in vitro-transcribed͞translated pulldown assays and in vivo coimmunoprecipitations revealed that TSAP6 associated with Nix, a proapoptotic Bcl-2-related protein and the Myt1 kinase, a negative regulator of the G 2͞M transition. Moreover, TSAP6 enhanced the susceptibility of cells to apoptosis and cooperated with Nix to exacerbate this effect. Cell-cycle studies indicated that TSAP6 could augment Myt1 activity. Overall, these data suggest that TSAP6 may act downstream to p53 to interface apoptosis and cell-cycle progression.

The bacterial cytolethal distending toxin (CDT) was previously shown to arrest the tumor-derived HeLa cell line in the G2-phase of the cell cycle through inactivation of CDK1, a cyclin-dependent kinase whose state of activation determines entry into mitosis. We have analysed the e ects induced in HeLa cells by CDT, in comparison to those induced by etoposide, a prototype anti-tumoral agent that triggers a G2 cell cycle checkpoint by inducing DNA damage. Both CDT and etoposide inhibit cell proliferation and induces the formation of enlarged mononucleated cells blocked in G2. In both cases, CDK1 from arrested cells could be reactivated both in vitro by dephosphorylation by recombinant Cdc25B phosphatase and in vivo by ca eine. However, the cell cycle arrest triggered by CDT, unlike etoposide, did not originate from DNA strand breaks as demonstrated in the single cell gel electrophoresis assay and by the absence of slowing down of S phase in synchronized cells. Together with additional observations on synchronized HeLa cells, our results suggest that CDT triggers a G2 cell cycle checkpoint that is initiated during DNA replication and that is independent of DNA damage.

CDC25B2, a protein tyrosine phosphatase closely related to the putative CDC25B oncogene, was identi®ed in a Burkitt lymphoma cDNA library. CDC25B2 di ers from CDC25B by a 14 residue insertion and a 41 residue deletion, which are both located in the amino-terminal region of the protein, upstream of the catalytic domain. Examination of the genomic sequence revealed that CDC25B1 (formerly B) and CDC25B2 are splice variants of the same gene. A third variant, CDC25B3, that carries both the 14 and the 41 residue sequences was also identi®ed in the same cDNA library. All three variants were detected in a panel of human primary culture and cell lines, although at di erent levels. In primary ®broblasts and in HeLa cells the CDC25B expression is cell cycle regulated, reaching a maximum in G 2 -phase. In vitro, CDC25B1 phosphatase is slightly more active than CDC25B2 and B3. However, episomal overexpression of the three CDC25B variants in ®ssion yeast reveals that in vivo, CDC25B2 is largely more active than either B1 or B3 (B24B34B1) both to complement a thermosensitive S pombe CDC25 activity and to act as a mitotic inducer. Alternative splicing of CDC25B may therefore contribute to the control of cell proliferation.

In eukaryotes the activity of CDK1 (CDC2), a cyclindependent kinase that initiates the structural changes that culminate in the segregation of chromosomes at mitosis, is regulated by the synergistic and opposing activities of a cascade of kinases and phosphatases. Dephosphorylation of threonine 14 and tyrosine 15 of CDK1 by the CDC25 phosphatases is a key step in the activation of the CDK1-cyclin B protein kinase. Little is currently known about the role and the regulation of CDC25B. Here we report in vitro and in vivo data that indicate that CDC25B is degraded by the proteasome. This degradation is dependent upon phosphorylation by the CDK1-cyclin A complex but not by CDK1-cyclin B. These results indicate that CDK1-cyclin A phosphorylation targets CDC25B for degradation and that this might be an important component of cell cycle regulation at the G 2 /M transition.The eukaryotic cell cycle is controlled by a family of cyclindependent kinases that regulate its key transitions. The precise timing of the activation of these enzymes is a central issue of cell cycle regulation. A major regulatory mechanism is provided by the wee1/mik1-and myt1-dependent phosphorylation on threonine 14 and tyrosine 15 of CDK1 (cdc2). This inhibitory phosphorylation keeps the kinase inactive until it is dephosphorylated by the dual specificity CDC25 phosphatase at the G 2 /M transition (1). In human cells, three homologues of CDC25 called CDC25A, CDC25B, and CDC25C have been identified (2-4). In HeLa cells, expression of CDC25B is low throughout the cell cycle with an increase in G 2 , CDC25C is also predominantly expressed in G 2 (2, 3), and CDC25A is abundant both at the mRNA and protein levels in late G 1 (5). Phosphorylation of CDC25C by CDK1-cyclin B was shown and proposed to be part of the self-amplification mechanism of CDK1-cyclin B at mitosis (6). CDK2-cyclin E-dependent phosphorylation of CDC25A was also demonstrated, indicating that a similar feedback loop might regulate the progression in S phase (7). The mechanisms that regulate CDC25B activity and the precise role of that phosphatase and its splicing variants (8) in the control of entry into mitosis remain unclear.Here we report in vitro and in vivo evidences indicating that CDC25B is degraded by the proteasome and that this process is dependent on the phosphorylation by the CDK1-cyclin A kinase. We propose that the rapid degradation of CDC25B is an important regulatory mechanism that ensures the timely coordinated activation of CDK-cyclin complexes. MATERIALS AND METHODSProduction of Recombinant Proteins-The CDC25B coding sequence corresponding to the B1 splicing variant (8) was cloned in the pET14b (Novagen) vector. In vitro transcription and translation were performed using the TNT system (Promega) in the presence or the absence of [ 35 S]methionine. Sf9 insect cells were co-infected with recombinant baculovirus encoding for human CDK1 (CDC2) or CDK2 and cyclin B or cyclin A. Insect cell extracts were prepared as follows: cells were lysed with lysis buffer (10 mM Tris...

CDC25B phosphatases are essential regulators that control cyclin-dependent kinases activities at the entry into mitosis. In this study, we demonstrate that serine 146 is required for two crucial features of CDC25B1. It is essential for CDC25B1 to function as a mitotic inducer and to prevent CDC25B1 export from the nucleus. We also show that serine 146 is phosphorylated in vitro by CDK1-cyclin B. However, phosphorylation of CDC25B does not stimulate its phosphatase activity, and mutation of serine 146 had no effect on its catalytic activity. Serine 146 phosphorylation is proposed to be a key event in the regulation of the CDC25B function in the initiation of mammalian mitosis.