Multiple metalloproteinases process protransforming growth factor-alpha (proTGF-alpha)

Shedding of TNF-alpha requires a single cleavage event, whereas the ectodomain of proTGF-alpha is cleaved at N-proximal (N-terminal) and membrane proximal (C-terminal) sites to release mature TGF-alpha. Tumor necrosis factor-alpha converting enzyme (TACE) was shown to have a central role in the shedding of both factors. Here we show that cleavage of the proTGF-alpha C-terminal site, required for release of mature growth factor, is less sensitive to a panel of hydroxamates than TNF-alpha processing. Recombinant TACE cleaves TNF-alpha and N-terminal TGF-alpha peptides 50-fold more efficiently than the C-terminal TGF-alpha peptide. Moreover, fractionation of rat liver epithelial cell membranes yields two populations: one contains TACE and cleaves peptides corresponding to TNF-alpha and both proTGF-alpha processing sites, while the other lacks detectable TACE and cleaves only the C-terminal proTGF-alpha processing site. Activities in both fractions are inhibited by hydroxamates and EDTA but not by cysteine, aspartate, or serine protease inhibitors. Both membrane fractions also contain ADAM 10. ADAM 10 correctly cleaves peptides and a soluble form of precursor TGF-alpha (proTGFecto) at the N-terminal site but not the C-terminal site. However, the kinetics of N-terminal peptide cleavage by ADAM 10 are 90-fold less efficient than TACE. Our findings indicate that while TACE is an efficient proTGF-alpha N-terminal convertase, a different activity, distinguishable from TACE, exists that can process proTGF-alpha at the C-terminal site. A model that accounts for these findings and the requirement for TACE in TGF-alpha shedding is proposed.     

A role for a PDZ protein in the early secretory pathway for the targeting of proTGF-alpha to the cell surface

In general, plasma membrane integral proteins, such as the membrane-anchored growth factor proTGF-alpha, are assumed to be transported to the cell surface via a nonregulated, constitutive pathway. proTGF-alpha C-terminal mutants are retained in an early secretory compartment. Here, using a two-hybrid screen, we identify two TACIPs (proTGF-alpha cytoplasmic domain-interacting proteins) that contain PDZ domains and do not interact with proTGF-alpha C-terminal mutants. The binding specificity of one of them, TACIP18 (previously identified and named Syntenin or mda-9), coincides with that of the component that possibly mediates the normal trafficking of proTGF-alpha. TACIP18 colocalizes and interacts specifically with immature, intracellular forms of proTGF-alpha. Therefore, it appears that the interaction of TACIP18 with proTGF-alpha in the early secretory pathway is necessary for the targeting of the latter to the cell surface.     

TACE is required for the activation of the EGFR by TGF-alpha in tumors

The factors and mechanisms that transduce the intracellular signals sent upon activation of the receptor for the epidermal growth factor (EGFR) and related receptors are reasonably well understood and, in fact, are the targets of anti-tumor drugs. In contrast, less is known about the mechanisms implicated in sending the signals that activate these receptors. Here we show that when its proteolytic shedding is prevented, the transmembrane form of the transforming growth factor-alpha (proTGF-alpha) interacts with, but does not activate, the EGFR. Thus, shedding seems to control not only the availability of the soluble form of the growth factor (TGF-alpha) but also the activity of the transmembrane form. The activity of the protease responsible for the shedding of proTGF-alpha, tumor necrosis factor-alpha converting enzyme (TACE), is required for the activation of the EGFR in vivo and for the development of tumors in nude mice, indicating a crucial role of TACE in tumorigenesis. In agreement with this view, TACE is dramatically overexpressed in the majority of mammary tumors analyzed. Collectively, this evidence points to TACE as a promising target of anti-tumor therapy.     

Loss of ectodomain shedding due to mutations in the metalloprotease and cysteine-rich/disintegrin domains of the tumor necrosis factor-alpha converting enzyme (TACE)

Tumor necrosis factor-alpha converting enzyme (TACE), a multidomain protease essential for development and disease, releases the ectodomains from many transmembrane proteins in a regulated fashion. To understand the mechanism underlying the regulation of TACE activity, we sought to identify the cause of ectodomain shedding deficiencies in two mutated CHO sublines designated M1 and M2. Transfection of expression vectors for human and mouse TACE restored ectodomain shedding of TNF-alpha and TGF-alpha, suggesting that defects in the TACE gene contribute to the phenotype of M1 and M2 cells. The overall levels of endogenous TACE forms in M1 cells were significantly lower than those found in their parental cells, whereas only TACE zymogen, but not its mature form, was detectable in M2 cells. Molecular analyses suggested that M1 cells contained only one expressible TACE allele encoding an M435I point mutation in the catalytic center of the protease, and M2 cells produced two TACE variants with distinct point mutations in the catalytic domain (C225Y) and the cysteinerich/disintegrin domain (C600Y). Overexpression of the C225Y and C600Y TACE by transient transfection largely compensated for maturation defects in the variants but failed to restore TNF-alpha and TGF-alpha release in the shedding-defective CHO cell lines and fibroblasts derived from TACE-null mouse embryo. Further mutagenesis and functional analyses demonstrated that Cys(600) was absolutely essential for ectodomain shedding, suggesting that Cys(600), similar to Cys(225), participates in disulfide bonding, which is critical for both the processing and catalysis of TACE.     

α‐secretase mediated conversion of the amyloid precursor protein derived membrane stub C99 to C83 limits Aβ generation

The Swedish mutation within the amyloid precursor protein (APP) causes early‐onset Alzheimer’s disease due to increased cleavage of APP by BACE1. While β‐secretase shedding of Swedish APP (APPswe) largely results from an activity localized in the late secretory pathway, cleavage of wild‐type APP occurs mainly in endocytic compartments. However, we show that liberation of Aβ from APPswe is still dependent on functional internalization from the cell surface. Inspite the unchanged overall β‐secretase cleaved soluble APP released from APP_swe secretion, mutations of the APPswe internalization motif strongly reduced C99 levels and substantially decreased Aβ secretion. We point out that α‐secretase activity‐mediated conversion of C99 to C83 is the main cause of this Aβ reduction. Furthermore, we demonstrate that α‐secretase cleavage of C99 even contributes to the reduction of Aβ secretion of internalization deficient wild‐type APP. Therefore, inhibition of α‐secretase cleavage increased Aβ secretion through diminished conversion of C99 to C83 in APP695, APP695swe or C99 expressing cells.     

Impaired trafficking and activation of tumor necrosis factor-alpha-converting enzyme in cell mutants defective in protein ectodomain shedding

Protein ectodomain shedding is a specialized type of regulated proteolysis that releases the extracellular domain of transmembrane proteins. The metalloprotease disintegrin tumor necrosis factor-alpha-converting enzyme (TACE) has been convincingly shown to play a central role in ectodomain shedding, but despite its broad interest, very little is known about the mechanisms that regulate its activity. An analysis of the biosynthesis of TACE in mutant cell lines that have a gross defect in ectodomain shedding (M1 and M2) shows a defective removal of the prodomain that keeps TACE in an inactive form. Using LoVo, a cell line that lacks of active furin, and alpha1-Antitrypsin Portland, a protein inhibitor of proprotein convertases, we show that TACE is normally processed by furin and other proprotein convertases. The defect in M1 and M2 cells is due to a blockade of the exit of TACE from the endoplasmic reticulum. The processing of other zinc-dependent metalloproteases, previously suggested to participate in activated ectodomain shedding is normal in the mutant cells, indicating that the component mutated is highly specific for TACE. In summary, the characterization of shedding-defective somatic cell mutants unveils the existence of a specific mechanism that directs the proteolytic activation of TACE through the control of its exit from the ER.     

Tumor necrosis factor-alpha-converting enzyme (TACE/ADAM-17) mediates the ectodomain cleavage of intercellular adhesion molecule-1 (ICAM-1)

Ectodomain shedding has emerged as an important regulatory step in the function of transmembrane proteins. Intercellular adhesion molecule-1 (ICAM-1), an adhesion receptor that mediates inflammatory and immune responses, undergoes shedding in the presence of inflammatory mediators and phorbol 12-myristate 13-acetate (PMA). The shedding of ICAM-1 in ICAM-1-transfected 293 cells upon PMA stimulation and in endothelial cells upon tumor necrosis factor-alpha stimulation was blocked by metalloproteinase inhibitors, whereas serine protease inhibitors were ineffective. p-Aminophenylmercuric acetate, a mercuric compound that is known to activate matrix metalloproteinases, up-regulated ICAM-1 shedding. TIMP-3 (but not TIMP-1 or -2) effectively blocked cleavage. This profile suggests the involvement of the ADAM family of proteases in the cleavage of ICAM-1. The introduction of enzymatically active tumor necrosis factor-alpha-converting enzyme (TACE) into ICAM-1-expressing cells up-regulated cleavage. Small interfering RNA directed against TACE blocked ICAM-1 cleavage. ICAM-1 transfected into TACE-/- fibroblasts did not show increased shedding over constitutive levels in the presence of PMA, whereas cleavage did occur in ICAM-1-transfected TACE+/+ cells. These results indicate that ICAM-1 shedding is mediated by TACE. Blocking the shedding of ICAM-1 altered the cell adhesive function, as ICAM-1-mediated cell adhesion was up-regulated in the presence of TACE small interfering RNA and TIMP-3, but not TIMP-1. However, cleavage was found to occur at multiple sites within the stalk domain of ICAM-1, and numerous point mutations within the region did not affect cleavage, indicating that TACE-mediated cleavage of ICAM-1 may not be sequence-specific.     

The role of ADAM10 and ADAM17 in the ectodomain shedding of angiotensin converting enzyme and the amyloid precursor protein.

Numerous transmembrane proteins, including the blood pressure regulating angiotensin converting enzyme (ACE) and the Alzheimer's disease amyloid precursor protein (APP), are proteolytically shed from the plasma membrane by metalloproteases. We have used an antisense oligonucleotide (ASO) approach to delineate the role of ADAM10 and tumour necrosis factor-alpha converting enzyme (TACE; ADAM17) in the ectodomain shedding of ACE and APP from human SH-SY5Y cells. Although the ADAM10 ASO and TACE ASO significantly reduced (> 81%) their respective mRNA levels and reduced the alpha-secretase shedding of APP by 60% and 30%, respectively, neither ASO reduced the shedding of ACE. The mercurial compound 4-aminophenylmercuric acetate (APMA) stimulated the shedding of ACE but not of APP. The APMA-stimulated secretase cleaved ACE at the same Arg-Ser bond in the juxtamembrane stalk as the constitutive secretase but was more sensitive to inhibition by a hydroxamate-based compound. The APMA-activated shedding of ACE was not reduced by the ADAM10 or TACE ASOs. These results indicate that neither ADAM10 nor TACE are involved in the shedding of ACE and that APMA, which activates a distinct ACE secretase, is the first pharmacological agent to distinguish between the shedding of ACE and APP.     

Amyloid precursor-like protein 1 influences endocytosis and proteolytic processing of the amyloid precursor protein

Ectodomain shedding of the amyloid precursor protein (APP) is a key regulatory step in the generation of the Alzheimer disease amyloid beta peptide (Abeta). The molecular mechanisms underlying the control of APP shedding remain little understood but are in part dependent on the low density lipoprotein receptor-related protein (LRP), which is involved in APP endocytosis. Here, we show that the APP homolog APLP1 (amyloid precursor-like protein 1) influences APP shedding. In human embryonic kidney 293 cells expression of APLP1 strongly activated APP shedding by alpha-secretase and slightly reduced beta-secretase cleavage. As revealed by domain deletion analysis, the increase in APP shedding required the NPTY amino acid motif within the cytoplasmic domain of APLP1. This motif is conserved in APP and is essential for the endocytosis of APP and APLP1. Unrelated membrane proteins containing similar endocytic motifs did not affect APP shedding, showing that the increase in APP shedding was specific to APLP1. In LRP-deficient cells APLP1 no longer induced APP shedding, suggesting that in wild-type cells APLP1 interferes with the LRP-dependent endocytosis of APP and there by increases APP alpha-cleavage. In fact, an antibody uptake assay revealed that expression of APLP1 reduced the rate of APP endocytosis. In summary, our study provides a novel mechanism for APP shedding, in which APLP1 affects the endocytosis of APP and makes more APP available for alpha-secretase cleavage.     

Intracellularly generated amyloid-beta peptide counteracts the antiapoptotic function of its precursor protein and primes proapoptotic pathways for activation by other insults in neuroblastoma cells

Most mutations in amyloid precursor proteins (APPs) linked to early onset familial Alzheimer's disease (FAD) increase the production of amyloid-beta peptides ending at residue 42 (Abeta42), which are released from APP by beta- and gamma-secretase cleavage. Stably transfected cells expressing wild-type human APP (APP(WT)) were more resistant to apoptosis-inducing treatments than cells expressing FAD-mutant human APP (APP(FAD)). Preventing Abeta42 production with an M596I mutation (beta-), which blocks beta-secretase cleavage of APP, or by treatment with a gamma-secretase inhibitor increased the resistance of APP(FAD)-expressing cells to apoptosis. Exposing hAPP(FAD/beta-) cells to exogenous Abeta42 or conditioned medium from Abeta42-producing APP(FAD) cells did not diminish their resistance to apoptosis. Preventing APP from entering the distal secretory pathway, where most Abeta peptides are generated, by retaining APP in the endoplasmic reticulum (ER)/intermediate compartment (IC) increased the resistance of APP(FAD)-expressing cells to apoptosis and did not alter the resistance of APP(WT)-expressing cells. p53-mediated gene transactivation after apoptosis-inducing treatments was much stronger in APP(FAD) cells than in hAPP(WT) or hAPP(FAD/beta-) cells. In contrast, upon induction of ER stress, cells expressing APP(FAD), hAPP(FAD/beta-), or APP(WT) had comparable levels of glucose-regulated protein-78 mRNA, an unfolded protein response indicator. We conclude that Abeta, especially intracellular Abeta, counteracts the antiapoptotic function of its precursor protein and predisposes cells to p53-mediated, and possibly other, proapoptotic pathways.     

Characterization of TPM1 disrupted yeast cells indicates an involvement of tropomyosin in directed vesicular transport

Disruption of the yeast tropomyosin gene TPM1 results in the apparent loss of actin cables from the cytoskeleton (Liu, H., and A. Bretscher. 1989. Cell. 57:233-242). Here we show that TPM1 disrupted cells grow slowly, show heterogeneity in cell size, have delocalized deposition of chitin, and mate poorly because of defects in both shmooing and cell fusion. The transit time of alpha-factor induced a-agglutinin secretion to the cell surface is longer than in isogenic wild-type strains, and some of the protein is mislocalized. Many of the TPM1-deleted cells contain abundant vesicles, similar in morphology to late secretory vesicles, but without an abnormal accumulation of intermediates in the delivery of either carboxypeptidase Y to the vacuole or invertase to the cell surface. Combinations of the TPM1 disruption with sec13 or sec18 mutations, which affect early steps in the secretory pathway, block vesicle accumulation, while combinations with sec1, sec4 or sec6 mutations, which affect a late step in the secretory pathway, have no effect on the vesicle accumulation. The phenotype of the TPM1 disrupted cells is very similar to that of a conditional mutation in the MYO2 gene, which encodes a myosin-like protein (Johnston, G. C., J. A. Prendergast, and R. A. Singer. 1991. J. Cell Biol. 113:539-551). The myo2-66 conditional mutation shows synthetic lethality with the TPM1 disruption, indicating that the MYO2 and TPM1 gene products may be involved in the same, or parallel function. We conclude that tropomyosin, and by inference actin cables, may facilitate directed vesicular transport of components to the correct location on the cell surface.     

Tumor necrosis factor-alpha-converting enzyme controls surface expression of c-Kit and survival of embryonic stem cell-derived mast cells

Transmembrane metalloproteinases of the disintegrin and metalloproteinase (ADAM) family control cell signaling interactions via hydrolysis of protein extracellular domains. Prior work has shown that the receptor tyrosine kinase, c-Kit (CD117), is essential for mast cell survival and that serum levels of c-Kit increase in proliferative mast cell disorders, suggesting the existence of c-Kit shedding pathways in mast cells. In the present work, we report that tumor necrosis factor alpha-converting enzyme (TACE; ADAM-17) mediates shedding of c-Kit. Stimulation of transfected cells with phorbol 12-myristate 13-acetate (PMA) induced metalloproteinase-mediated release of c-Kit ectodomain, which increased further upon TACE overexpression. By contrast, TACE-deficient fibroblasts did not demonstrate inducible release, thus identifying TACE as the metalloproteinase primarily responsible for PMA-induced c-Kit shedding. Surface expression of c-Kit by the human mast cell-1 line decreased upon phorbol-induced shedding, which involved metalloproteinase activity susceptible to inhibition by tissue inhibitor of metalloproteinase (TIMP)-3. To further explore the role of TACE in shedding of c-Kit from mast cells, we compared the behavior of mast cells derived from murine embryonic stem cells. In these studies, PMA decreased surface c-Kit levels on mast cells expressing wild-type (+/+) TACE but not on those expressing an inactive mutant (DeltaZn/DeltaZn), confirming the role of TACE in PMA-induced c-Kit shedding. Compared with TACE(+/+) cells, TACE(DeltaZn/DeltaZn) mast cells also demonstrated decreased constitutive shedding and increased basal surface expression of c-Kit, with diminished apoptosis in response to c-Kit ligand deprivation. These data suggest that TACE controls mast cell survival by regulating shedding and surface expression of c-Kit.     

Shedding of the amyloid precursor protein-like protein APLP2 by disintegrin-metalloproteinases

Cleavage of the amyloid precursor protein (APP) within the amyloid-beta (Abeta) sequence by the alpha-secretase prevents the formation of toxic Abeta peptides. It has been shown that the disintegrin-metalloproteinases ADAM10 and TACE (ADAM17) act as alpha-secretases and stimulate the generation of a soluble neuroprotective fragment of APP, APPsalpha. Here we demonstrate that the related APP-like protein 2 (APLP2), which has been shown to be essential for development and survival of mice, is also a substrate for both proteinases. Overexpression of either ADAM10 or TACE in HEK293 cells increased the release of neurotrophic soluble APLP2 severalfold. The strongest inhibition of APLP2 shedding in neuroblastoma cells was observed with an ADAM10-preferring inhibitor. Transgenic mice with neuron-specific overexpression of ADAM10 showed significantly increased levels of soluble APLP2 and its C-terminal fragments. To elucidate a possible regulatory mechanism of APLP2 shedding in the neuronal context, we examined retinoic acid-induced differentiation of neuroblastoma cells. Retinoic acid treatment of two neuroblastoma cell lines upregulated the expression of both APLP2 and ADAM10, thus leading to an increased release of soluble APLP2.     

Cleavage of Alzheimer's amyloid precursor protein by alpha-secretase occurs at the surface of neuronal cells.

The amyloid precursor protein (APP) is proteolytically processed predominantly by alpha-secretase to release the ectodomain (sAPPalpha). In this study, we have addressed the cellular location of the constitutive alpha-secretase cleavage of endogenous APP in a neuronal cell line. Incubation of the neuroblastoma cell line IMR32 at 20 degrees C prevented the secretion into the medium of soluble wild-type APP cleaved by alpha-secretase as revealed by both immunoelectrophoretic blot analysis with a site-specific antibody and immunoprecipitation following metabolic labeling of the cells. No sAPPalpha was detected in the cell lysates following incubation of the cells at 20 degrees C, indicating that alpha-secretase does not cleave APP in the secretory pathway prior to or within the trans-Golgi network. Parallel studies using an antibody that recognizes specifically the neoepitope revealed on soluble APP cleaved by beta-secretase indicated that this enzyme was acting intracellularly. alpha-Secretase is a zinc metalloproteinase susceptible to inhibition by hydroxamate-based compounds such as batimastat [Parvathy, S., et al. (1998) Biochemistry 37, 1680-1685]. Incubation of the cells with a cell-impermeant, biotinylated hydroxamate inhibitor inhibited the release of sAPPalpha by >92%, indicating that alpha-secretase is cleaving APP almost exclusively at the cell surface. The observation that alpha-secretase cleaves APP at the cell surface, while beta-secretase can act earlier in the secretory pathway within the neuronal cell line indicates that there must be strict control mechanisms in place to ensure that APP is normally cleaved primarily by alpha-secretase in the nonamyloidogenic pathway to produce the neuroprotective sAPPalpha.     

Metalloprotease-dependent protransforming growth factor-alpha ectodomain shedding in the absence of tumor necrosis factor-alpha-converting enzyme

Zinc-dependent metalloproteases can mediate the shedding of the extracellular domain of many unrelated transmembrane proteins from the cell surface. In most instances, this process, also known as ectodomain shedding, is regulated via protein kinase C (PKC). The tumor necrosis factor alpha-converting enzyme (TACE) was the first protease involved in regulated protein ectodomain shedding identified. Although TACE belongs to the family of metalloprotease-disintegrins, few members of this family have been shown to participate in regulated ectodomain shedding. In fact, the phenotype of tace-/- cells and that of Chinese hamster ovary cell mutants defective in ectodomain shedding points to the existence of a common PKC-activated ectodomain shedding system, whose proteolytic component is TACE, that acts on a variety of transmembrane proteins. Examples of these proteins include the Alzheimer's disease-related protein beta-amyloid precursor protein (betaAPP) and the transmembrane growth factors protransforming growth factor-alpha (pro-TGF-alpha) and, as shown in this report, proheparin-binding epidermal growth factor-like growth factor (pro-HB-EGF). Here we show that the mercurial compound 4-aminophenylmercuric acetate (APMA), frequently used to activate in vitro recombinant matrix metalloproteases, is an activator of the shedding of betaAPP, pro-HB-EGF, and pro-TGF-alpha. Treatment of tace-/- cells or Chinese hamster ovary shedding-defective mutants with APMA activates the cleavage of pro-TGF-alpha but not that of pro-HB-EGF or betaAPP, indicating that APMA activates TACE and also a previously unacknowledged proteolytic activity specific for pro-TGF-alpha. Characterization of this proteolytic activity indicates that it acts on pro-TGF-alpha located at the cell surface and that it is a metalloprotease active in cells defective in furin activity. In summary, treatment of shedding-defective cell lines with APMA unveils the existence of a metalloprotease activity alternative to TACE with the ability to specifically shed the ectodomain of pro-TGF-alpha.     

Protein kinase C regulation of intracellular and cell surface amyloid precursor protein (APP) cleavage in CHO695 cells

Cleavage of amyloid precursor protein (APP) by beta-secretase generates beta-amyloid (Abeta), the major component of senile plaques in Alzheimer's disease. Cleavage of APP by alpha-secretase prevents Abeta formation, producing nonamyloidogenic secreted APPs products. PKC-regulated APP alpha-secretase cleavage has been shown to involve tumor necrosis factor alpha (TNF-alpha) converting enzyme (TACE). To determine the location of APP cleavage, we examined PKC-regulated APPs secretion by examining cell surface versus intracellular APP in CHO cells stably expressing APP(695) (CHO695). We demonstrate that PKC regulates cell surface and intracellular APP cleavage. The majority of secreted APPs originates from the intracellular compartment, and PKC does not cause an increase in APP trafficking to the cell surface for cleavage. Therefore, intracellular APP regulated by PKC must be cleaved at an intracellular site. Experiments utilizing Brefeldin A suggest APP cleavage occurs at the Golgi or late in the secretory pathway. Experiments using TAPI, an inhibitor of TACE, demonstrate PKC-regulated APPs secretion from the cell surface is inhibited after pretreatment with TAPI, and APPs secretion from the intracellular pool is partially inhibited after pretreatment with TAPI. These findings suggest PKC-regulated APP cleavage occurs at multiple locations within the cell and both events appear to involve TACE.     

Mislocalization of CDK11/PITSLRE, a regulator of the G2/M phase of the cell cycle, in Alzheimer disease

Post-mitotic neurons are typically terminally differentiated and in a quiescent status. However, in Alzheimer disease (AD), many neurons display ectopic re-expression of cell cycle-related proteins. Cyclin-dependent kinase 11 (CDK11) mRNA produces a 110-kDa protein (CDK11(p110)) throughout the cell cycle, a 58-kDa protein (CDK11(p58)) that is specifically translated from an internal ribosome entry site and expressed only in the G(2)/M phase of the cell cycle, and a 46-kDa protein (CDK11(p46)) that is considered to be apoptosis specific. CDK11 is required for sister chromatid cohesion and the completion of mitosis. In this study, we found that the expression patterns of CDK11 vary such that cytoplasmic CDK11 is increased in AD cellular processes, compared to a pronounced nuclear expression pattern in most controls. We also investigated the effect of amyloid precursor protein (APP) on CDK11 expression in vitro by using M17 cells overexpressing wild-type APP and APP Swedish mutant phenotype and found increased CDK11 expression compared to empty vector. In addition, amyloid-β(25-35) resulted in increased CDK11 in M17 cells. These data suggest that CDK11 may play a vital role in cell cycle re-entry in AD neurons in an APP-dependent manner, thus presenting an intriguing novel function of the APP signaling pathway in AD.     

Genetic studies with the fission yeast Schizosaccharomyces pombe suggest involvement of wee1, ppa2, and rad24 in induction of cell cycle arrest by human immunodeficiency virus type 1 Vpr

Accessory protein Vpr of human immunodeficiency virus type 1 (HIV-1) arrests cell cycling at G(2)/M phase in human and simian cells. Recently, it has been shown that Vpr also causes cell cycle arrest in the fission yeast Schizosaccharomyces pombe, which shares the cell cycle regulatory mechanisms with higher eukaryotes including humans. In this study, in order to identify host cellular factors involved in Vpr-induced cell cycle arrest, the ability of Vpr to cause elongated cellular morphology (cdc phenotype) typical of G(2)/M cell cycle arrest in wild-type and various mutant strains of S. pombe was examined. Our results indicated that Vpr caused the cdc phenotype in wild-type S. pombe as well as in strains carrying mutations, such as the cdc2-3w, Deltacdc25, rad1-1, Deltachk1, Deltamik1, and Deltappa1 strains. However, other mutants, such as the cdc2-1w, Deltawee1, Deltappa2, and Deltarad24 strains, failed to show a distinct cdc phenotype in response to Vpr expression. Results of these genetic studies suggested that Wee1, Ppa2, and Rad24 might be required for induction of cell cycle arrest by HIV-1 Vpr. Cell proliferation was inhibited by Vpr expression in all of the strains examined including the ones that did not show the cdc phenotype. The results supported the previously suggested possibility that Vpr affects the cell cycle and cell proliferation through different pathways.     

The beta-amyloid precursor protein is not processed by the regulated secretory pathway.

The beta-amyloid peptide is derived from a larger membrane bound protein and accumulates as amyloid in Alzheimer's diseased brains. beta-amyloid precursor protein (beta APP) proteolytically processed during constitutive secretion cannot be a source of deposited amyloid because this processing results in cleavage within the amyloidogenic peptide. To see if other secretory pathways could be responsible for generating potentially amyloidogenic molecules we tested the possibility that beta APP is targeted to the regulated secretory pathway. Stable AtT20 cell lines expressing exogenous human beta APP were genetically engineered. These cells were labeled with [35S]-methionine, and chased in the presence or absence of secretagogue. The beta APP both inside the cells and released from the cells was analyzed by immunoprecipitation and gel analysis. Quantitation of autoradiograms showed that virtually all of the synthesized beta APP was secreted by the constitutive pathway, and that no detectable (less than 1%) beta APP was targeted to the regulated secretory pathway.     

Regulated cell surface pro-EGF ectodomain shedding is a zinc metalloprotease-dependent process

Epidermal growth factor receptor (EGFR) ligands are synthesized as type I membrane protein precursors exposed at the cell surface. Shedding of the ectodomain of these proteins is the way cells regulate the equilibrium between cell-associated and diffusible forms of these growth factors. Whereas the regulated shedding of transforming growth factor-alpha, HB-EGF, and amphiregulin precursors have been clearly established, regulation of full-length pro-EGF shedding has not been clearly demonstrated. Here, using both wild-type and M2 mutant CHO-K1 as well as HeLa cell lines transiently transfected with epitope-tagged rat pro-EGF expression plasmid, we demonstrate that these cells synthesize EGF as a high molecular weight membrane-associated precursor glycoprotein expressed at the cell surface. All cell lines are able to release the entire ectodomain of pro-EGF in the extracellular medium following juxtamembrane cleavage of the precursor once it is present at the cell surface. More significantly we clearly established that CHO-M2 and HeLa cells only constitutively release low levels of pro-EGF. This shedding is a regulated phenomenon in wild-type CHO cells where it can be induced by different agents such as phorbol 12-myristate 13-acetate (PMA), pervanadate, and serum but not by calcium ionophores. Using specific inhibitors as well as protein kinase C (PKC) depletion, PMA stimulation was shown to be completely dependent on PKC activation whereas pervanadate and serum stimulation were not. Regulated ectodomain shedding involves the activity of a zinc metalloprotease as determined by inhibition with phenantrolin and TAPI-2 and by the results obtained with the CHO-M2 shedding defective mutant cell line. Comparison of the ability of CHO and HeLa cell lines to shed pro-EGF and pro-TNF-alpha upon stimulation greatly suggests that TACE (ADAM 17) may not be the ectoprotease involved in the secretion of pro-EGF ectodomain and that this protease, which remains to be identified, shows a restricted cellular expression pattern.