Dual processing of FAT1 cadherin protein by human melanoma cells generates distinct protein products

The giant cadherin FAT1 is one of four vertebrate orthologues of the Drosophila tumor suppressor fat. It engages in several functions, including cell polarity and migration, and in Hippo signaling during development. Homozygous deletions in oral cancer suggest that FAT1 may play a tumor suppressor role, although overexpression of FAT1 has been reported in some other cancers. Here we show using Northern blotting that human melanoma cell lines variably but universally express FAT1 and less commonly FAT2, FAT3, and FAT4. Both normal melanocytes and keratinocytes also express comparable FAT1 mRNA relative to melanoma cells. Analysis of the protein processing of FAT1 in keratinocytes revealed that, like Drosophila FAT, human FAT1 is cleaved into a non-covalent heterodimer before achieving cell surface expression. The use of inhibitors also established that such cleavage requires the proprotein convertase furin. However, in melanoma cells, the non-cleaved proform of FAT1 is also expressed at the cell surface together with the furin-cleaved heterodimer. Moreover, furin-independent processing generates a potentially functional proteolytic product in melanoma cells, a persistent 65-kDa membrane-bound cytoplasmic fragment no longer in association with the extracellular fragment. In vitro localization studies of FAT1 showed that melanoma cells display high levels of cytosolic FAT1 protein, whereas keratinocytes, despite comparable FAT1 expression levels, exhibited mainly cell-cell junctional staining. Such differences in protein distribution appear to reconcile with the different protein products generated by dual FAT1 processing. We suggest that the uncleaved FAT1 could promote altered signaling, and the novel products of alternate processing provide a dominant negative function in melanoma.     

Apoptin T108 phosphorylation is not required for its tumor-specific nuclear localization but partially affects its apoptotic activity

Apoptin, a chicken anemia virus-encoded protein, induces apoptosis in human tumor cells but not in normal cells. In addition, Apoptin also exhibits tumor-specific nuclear localization and tumor-specific phosphorylation on threonine 108 (T108). Here, we studied the effects of T108 phosphorylation on the tumor-specific nuclear localization and apoptotic activity of Apoptin. We first showed that a hemagglutinin (HA)-tagged Apoptin, but not the green fluorescent protein-fused Apoptin used in many previous studies, exhibited the same intracellular distribution pattern as native Apoptin. We then made and analyzed an HA-Apoptin mutant with its T108 phosphorylation site abolished. We found that Apoptin T108 phosphorylation is not required for its tumor-specific nuclear localization and abolishing the T108 phosphorylation of Apoptin does affect its apoptotic activity in tumor cells but only partially. Our results support the previous finding that Apoptin contains two distinct apoptosis domains located separately at the N- and C-terminal regions and suggest that the T108 phosphorylation may only be required for the apoptotic activity mediated through the C-terminal apoptosis domain.     

p120 serine and threonine phosphorylation is controlled by multiple ligand-receptor pathways but not cadherin ligation

p120-catenin (p120) regulates cadherin turnover and is required for cadherin stability. Extensive and dynamic phosphorylation on tyrosine, serine and threonine residues in the N-terminal regulatory domain has been postulated to regulate p120 function, possibly through modulation of the efficiency of p120/cadherin interaction. Here we have utilized novel phospho-specific monoclonal antibodies to four major p120 serine and threonine phosphorylation sites to monitor individual phosphorylation events and their consequences. Surprisingly, membrane-localization and not cadherin interaction is the main determinant in p120 serine and threonine phosphorylation and dephosphorylation. Furthermore, the phospho-status of these four residues had no obvious effect on p120's role in cadherin complex stabilization or cell-cell adhesion. Interestingly, dephosphorylation was dramatically induced by PKC activation, but PKC-independent pathways were also evident. The data suggest that p120 dephosphorylation at these sites is modulated by multiple cell surface receptors primarily through PKC-dependent pathways, but these changes do not seem to reduce p120/cadherin affinity.     

Atheroma development in apolipoprotein E-null mice is not regulated by phosphorylation of p27(Kip1) on threonine 187

Excessive cellular proliferation is thought to contribute to neointimal lesion development during atherosclerosis and restenosis after angioplasty. Inhibition of cyclin-dependent kinase (CDK) activity by p27 inhibits mammalian cell growth. Mounting evidence indicates that p27 negatively regulates neointimal thickening in animal models of restenosis and atherosclerosis, and its expression in human neointimal lesions is consistent with such a protective role. Cell cycle progression is facilitated by cyclinE/CDK2-dependent phosphorylation of p27 on threonine 187 (T187) during late G1. The purpose of this study was to assess whether this phosphorylation event plays a role during atherosclerosis. To this end, we generated apolipoprotein E-null mice with both p27 alleles replaced by a mutated form non-phosphorylatable at T187 (apoE-/-p27T187A mice) and investigated the kinetics of atheroma development in these animals compared to apoE-/- controls with an intact p27 gene. Fat feeding resulted in comparable level of hypercholesterolemia in both groups of mice. Surprisingly, aortic p27 expression was not increased in fat-fed apoE-/-p27T187A mice compared with apoE-/- controls. Moreover, atheroma size, lesion cellularity, proliferation, and apoptotic rates were undistinguishable in both groups of fat-fed mice. Thus, in contrast to previous studies that highlight the importance of p27 phosphorylation at T187 on the control of p27 expression and function in different tissues and pathophysiological scenarios, our findings demonstrate that this phosphorylation event is not implicated in the control of aortic p27 expression and atheroma progression in hypercholesterolemic mice.     

Asn229 in the third helix of VPAC1 receptor is essential for receptor activation but not for receptor phosphorylation and internalization: comparison with Asn216 in VPAC2 receptor

After stimulation with agonist, G protein coupled receptors (GPCR) undergo conformational changes that allow activation of G proteins to transduce the signal, followed by phosphorylation by kinases and arrestin binding to promote receptor internalization. Actual paradigm, based on a study of GPCR-A/rhodopsin family, suggests that a network of interactions between conserved residues located in transmembrane (TM) domains (mainly TM3, TM6 and TM7) is involved in the molecular switch leading to GPCR activation.

We evaluated in CHO cells expressing the VPAC₁ receptor the role of the third transmembrane helix in agonist signalling by point mutation into Ala of the residues highly conserved in the secretin-family of receptors: Y²²⁴, N²²⁹, F²³⁰, W²³², E²³⁶, G²³⁷, Y²³⁹, L²⁴⁰. N²²⁹A VPAC₁ mutant was characterized by a decrease in both potency and efficacy of VIP stimulated adenylate cyclase activity, by the absence of agonist stimulated [Ca²⁺]_i increase, by a preserved receptor recognition of agonists and antagonist and by a preserved sensitivity to GTP suggesting the importance of that residue for efficient G protein activation. N²²⁹D mutant was not expressed at the membrane, and the N²²⁹Q with a conserved mutation was less affected than the A mutant. Agonist stimulated phosphorylation and internalization of N²²⁹A and N²²⁹Q VPAC₁ were unaffected. However, the re-expression of internalized mutant receptors, but not that of the wild type receptor, was rapidly reversed after VIP washing. Receptor phosphorylation, internalization and re-expression may be thus dissociated from G protein activation and linked to another active conformation that may influence its trafficking.

Mutation of that conserved amino acid in VPAC₂ could be investigated only by a conservative mutation (N²¹⁶Q) and led to a receptor with a low VIP stimulation of adenylate cyclase, receptor phosphorylation and internalization. This indicated the importance of the conserved N residue in the TM3 of that family of receptors.     

The tumor suppressor protein DLC1 is regulated by PKD-mediated GAP domain phosphorylation

Deleted in liver cancer 1 (DLC1) is a tumor suppressor protein that is frequently downregulated in various tumor types. DLC1 contains a Rho GTPase activating protein (GAP) domain that appears to be required for its tumor suppressive functions. Little is known about the molecular mechanisms that regulate DLC1. By mass spectrometry we have mapped a novel phosphorylation site within the DLC1 GAP domain on serine 807. Using a phospho-S807-specific antibody, our results identify protein kinase D (PKD) to phosphorylate this site in DLC1 in intact cells. Although phosphorylation on serine 807 did not directly impact on in vitro GAP activity, a DLC1 serine-to-alanine exchange mutant inhibited colony formation more potently than the wild type protein. Our results thus show that PKD-mediated phosphorylation of DLC1 on serine 807 negatively regulates DLC1 cellular function.     

Stress-induced degradation of the photosynthetic apparatus is accompanied by changes in thylakoid protein turnover and phosphorylation

Development of chlorosis and loss of PSII were compared in young spinach plants suffering under a combined magnesium and sulphur deficiency. Loss of chlorophyll could be detected already after the first week of deficiency and preceded any permanent functional inhibition of PSII as detected by changes in the chlorophyll fluorescence parameter F_v/F_m. A substantial decrease in F_v/F_m was observed only after the second week of deficiency. After 4 weeks, the plants had lost about 70% of their original chlorophyll content, but fluorescence data indicated that 80% of the existing PSII centers were still capable of initiating photosynthetic electron transport. The degradation of the photosynthetic apparatus without loss of PSII activity was due to changes in protein turnover, especially of the PSII D1 reaction center protein. Already by day 7 of deficiency, a 1.4-fold increase in D1 protein synthesis was observed measured as incorporation of ¹⁴C-leucine. Immunological determination by western-blotting did not reveal a change in D1 protein content. Thus, D1 protein was also degraded more rapidly. The increased turnover was high enough to prevent any loss or inhibition of PSII. After 3 weeks, D1 protein synthesis on a chlorophyll basis was further increased by a factor of 2. However, this was not enough to prevent a net loss of D1 protein of about 70%. Immunological determination revealed that together with the D1 protein also other polypeptides of PSII became degraded. This process prevented a large accumulation of photo-inactivated PSII centers. However, it initiated the breakdown of the other thylakoid proteins, especially of LHCII, resulting in the observed chlorosis. Together with the change in protein turnover and stability, a characteristic change in thylakoid protein phosphorylation was observed. In the deficient plants steady state phosphorylation of both LHCII and PSII proteins was increased in the dark. In the light phosphorylation of PSII proteins was stimulated and after 3 weeks of deficiency was even higher in the deficient leaves than in the control plants. In contrast, the phosphorylation level of LHCII decreased in the light and could hardly be detected after 3 weeks of deficiency. Phosphorylation of the reaction center polypeptides presumably increased their stability against proteolytic attack, whereas phosphorylated LHCII seems to be the substrate for proteolysis.     

Phytochrome Kinase Substrate 4 is phosphorylated by the phototropin 1 photoreceptor

Phototropism allows plants to redirect their growth towards the light to optimize photosynthesis under reduced light conditions. Phototropin 1 (phot1) is the primary low blue light-sensing receptor triggering phototropism in Arabidopsis. Light-induced autophosphorylation of phot1, an AGC-class protein kinase, constitutes an essential step for phototropism. However, apart from the receptor itself, substrates of phot1 kinase activity are less clearly established. Phototropism is also influenced by the cryptochromes and phytochromes photoreceptors that do not provide directional information but influence the process through incompletely characterized mechanisms. Here, we show that Phytochrome Kinase Substrate 4 (PKS4), a known element of phot1 signalling, is a substrate of phot1 kinase activity in vitro that is phosphorylated in a phot1-dependent manner in vivo. PKS4 phosphorylation is transient and regulated by a type 2-protein phosphatase. Moreover, phytochromes repress the accumulation of the light-induced phosphorylated form of PKS4 showing a convergence of photoreceptor activity on this signalling element. Our physiological analyses suggest that PKS4 phosphorylation is not essential for phototropism but is part of a negative feedback mechanism.     

Degradation of FAT10 by the 26S proteasome is independent of ubiquitylation but relies on NUB1L

The ubiquitin-like modifier FAT10 targets proteins for degradation by the proteasome, a process accelerated by the UBL-UBA domain protein NEDD8 ultimate buster 1-long. Here, we show that FAT10-mediated degradation occurs independently of poly-ubiquitylation as purified 26S proteasome readily degraded FAT10-dihydrofolate reductase (DHFR) but not ubiquitin-DHFR in vitro. Interestingly, the 26S proteasome could only degrade FAT10-DHFR when NUB1L was present. Knock-down of NUB1L attenuated the degradation of FAT10-DHFR in intact cells suggesting that NUB1L determines the degradation rate of FAT10-linked proteins. In conclusion, our data establish FAT10 as a ubiquitin-independent but NUB1L-dependent targeting signal for proteasomal degradation.     

Spinophilin is phosphorylated by Ca2+/calmodulin-dependent protein kinase II resulting in regulation of its binding to F-actin

Spinophilin is a protein phosphatase-1- and actin-binding protein that modulates excitatory synaptic transmission and dendritic spine morphology. We have recently shown that the interaction of spinophilin with the actin cytoskeleton depends upon phosphorylation by protein kinase A. We have now found that spinophilin is phosphorylated by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in neurons. Ca(2+)/calmodulin-dependent protein kinase II, located within the post-synaptic density of dendritic spines, is known to play a role in synaptic plasticity and is ideally positioned to regulate spinophilin. Using tryptic phosphopeptide mapping, site-directed mutagenesis and microsequencing analysis, we identified two sites of CaMKII phosphorylation (Ser-100 and Ser-116) within the actin-binding domain of spinophilin. Phosphorylation by CaMKII reduced the affinity of spinophilin for F-actin. In neurons, phosphorylation at Ser-100 by CaMKII was Ca(2+) dependent and was associated with an enrichment of spinophilin in the synaptic plasma membrane fraction. These results indicate that spinophilin is phosphorylated by multiple kinases in vivo and that differential phosphorylation may target spinophilin to specific locations within dendritic spines.     

Nuclear protein import, but not mRNA export, is defective in all Saccharomyces cerevisiae mutants that produce temperature-sensitive forms of the Ran GTPase homologue Gsp1p

A series of ts mutations in the GSP1 gene of Saccharomyces cerevisiae was isolated by error-prone PCR. A total of 25 ts gsp1 strains was obtained. Each of these mutants showed between one and seven different amino acid alterations. In several of these ts gsp1 strains, the same amino acid residues in Gsp1p were repeatedly mutated, indicating that our screen for ts gsp1 mutations was saturating. All of the ts gsp1 strains isolated had a defect in nuclear protein import, but only 16 of the 25 ts gsp1 strains had a defect in mRNA export. Thus, Gsp1p is suggested to be directly involved in nuclear protein import, but not in mRNA export. Following release from α-factor arrest, 11 of the ts gsp1 mutants arrested in G1; the remainder did not show any specific cell-cycle arrest, at 37° C, the nonpermissive temperature. While the mutants that are defective in both mRNA export and protein import have a tendency to arrest in G1, there was no clear correlation between the cell cycle phenotype and the defects in mRNA export and nuclear protein import. Based on this, we assume that Ran/Gsp1p GTPase regulates the cell cycle and the nucleus/cytosol exchange of macromolecules through interactions with effectors that were independent of each other, and are differentially affected by mutation. Received: 30 June 1997 / Accepted: 23 October 1997     

Inhibition of CO2-fixation and its effect on the activity of Photosystem II,on D1-protein synthesis and phosphorylation

To study the effects of limitations in the Calvin-cycle on Photosystem (PS) II function and on its repair by D1-protein turnover, glycerinaldehyde (DLGA) was applied to 1 h dark-adapted pea leaves via the petiole. The application resulted in a 90% inhibition of photosynthetic oxygen evolution after 90 min illumination at either 120 or 500 µmol m^–2 s^–1. In the control leaves an increase of light-dependent oxygen production to 147 and 171% was observed after 90 min illumination. According to chlorophyll fluorescence quenching analysis the inhibition of photosynthetic electron transport by DLGA led to a substantial increase in the reduction state of the primary quinone acceptor of PS II, QA, and to a rise in membrane energetisation. However, PS II functionality was hardly affected by DLGA at the low light intensity as indicated by the constant high yield of variable fluorescence, Fv/Fm. Only at 500 µmol m^–2 s^–1 a 15% loss of Fv/Fm was observed in the presence of DLGA indicating that inactivated PS II centres had accumulated. The control leaves also showed a slight loss of Fv/Fm which did not affect photosynthetic electron transport due to a faster reoxidation of QA. The relative stability of PS II function in the presence of DLGA could not be ascribed to an increased repair by the rapid turnover of the D1-protein. Radioactive pulse-labelling studies with [14C] leucine in combination with immunological determination of the protein content revealed that both synthesis and degradation of the protein were inhibited in DLGA-treated leaves whereas in the control leaves a stimulation of D1-protein turnover was observed. The changes of D1-protein turnover could be explained by differences in the occupancy state of the QB-binding niche. A relation between the phosphorylation status of the PS II polypeptides and the turnover of the D1-protein could not be established. As shown by radioactive labelling with [32P]i, addition of DLGA led to an increase in the phosphorylation level of the PS II polypeptides D1 and D2 at the low light intensity when compared to the non-treated control. At the higher light intensity the phosphorylation level of the PS II polypeptides in control and DLGA-treated leaves were identical in spite of the substantial differences in D1-protein turnover.     

Sec61 channel subunit Sbh1/Sec61β promotes ER translocation of proteins with suboptimal targeting sequences and is fine-tuned by phosphorylation

The highly conserved endoplasmic reticulum (ER) protein translocation channel contains one nonessential subunit, Sec61β/Sbh1, whose function is poorly understood so far. Its intrinsically unstructured cytosolic domain makes transient contact with ER-targeting sequences in the cytosolic channel vestibule and contains multiple phosphorylation sites suggesting a potential for regulating ER protein import. In a microscopic screen, we show that 12% of a GFP-tagged secretory protein library depends on Sbh1 for translocation into the ER. Sbh1-dependent proteins had targeting sequences with less pronounced hydrophobicity and often no charge bias or an inverse charge bias which reduces their insertion efficiency into the Sec61 channel. We determined that mutating two N-terminal, proline-flanked phosphorylation sites in the Sbh1 cytosolic domain to alanine phenocopied the temperature-sensitivity of a yeast strain lacking SBH1 and its ortholog SBH2. The phosphorylation site mutations reduced translocation into the ER of a subset of Sbh1-dependent proteins, including enzymes whose concentration in the ER lumen is critical for ER proteostasis. In addition, we found that ER import of these proteins depended on the activity of the phospho-S/T–specific proline isomerase Ess1 (PIN1 in mammals). We conclude that Sbh1 promotes ER translocation of substrates with suboptimal targeting sequences and that its activity can be regulated by a conformational change induced by N-terminal phosphorylation.     

Tyrosine phosphorylation translocates beta-catenin from cell-->cell interface to the cytoplasm, but does not significantly enhance the LEF-1-dependent transactivating function

beta-catenin plays an essential role in cells, not only as a cadherin-associated complex, but also as a signaling molecule in the nucleus. Tyrosine phosphorylation of beta-catenin has been shown to correlate with tumorigenesis, cell migration, and developmental processes. However, its exact effects on downstream targets in the nucleus are not yet clear. In this study, we used HCT-15 colon carcinoma and NIH 3T3 fibroblasts as models to investigate the effects of a phosphotyrosine phosphatase (PTPase) inhibitor on the localization of beta-catenin, the binding affinity to LEF-1 (Lymphoid Enhancer Factor), and on LEF-1-dependent transactivation function. Treatment with a PTPase inhibitor, pervanadate, increased the tyrosine phosphorylation of beta-catenin in a time-dependent manner and led to its relocation from cell-cell interfaces to the cytoplasm. This phosphorylation/dephosphorylation of beta-catenin does not require its presence at cell-cell interfaces. However, tyrosine phosphorylation of beta-catenin does not change its binding affinity to LEF-1 nor enhance cyclin D1 transactivation, a nuclear target of beta-catenin/LEF-1. This result suggests that tyrosine phosphorylation of beta-catenin has effects on the binding to cadherins in the cytoplasm but not on its LEF-1-dependent transactivating function in the nucleus.     

Direct interaction of DNA repair protein tyrosyl DNA phosphodiesterase 1 and the DNA ligase III catalytic domain is regulated by phosphorylation of its flexible N-terminus

Tyrosyl DNA phosphodiesterase 1 (TDP1) and DNA Ligase IIIα (LigIIIα) are key enzymes in single-strand break (SSB) repair. TDP1 removes 3′-tyrosine residues remaining after degradation of DNA topoisomerase (TOP) 1 cleavage complexes trapped by either DNA lesions or TOP1 inhibitors. It is not known how TDP1 is linked to subsequent processing and LigIIIα-catalyzed joining of the SSB. Here we define a direct interaction between the TDP1 catalytic domain and the LigIII DNA-binding domain (DBD) regulated by conformational changes in the unstructured TDP1 N-terminal region induced by phosphorylation and/or alterations in amino acid sequence. Full-length and N-terminally truncated TDP1 are more effective at correcting SSB repair defects in TDP1 null cells compared with full-length TDP1 with amino acid substitutions of an N-terminal serine residue phosphorylated in response to DNA damage. TDP1 forms a stable complex with LigIII_170–755, as well as full-length LigIIIα alone or in complex with the DNA repair scaffold protein XRCC1. Small-angle X-ray scattering and negative stain electron microscopy combined with mapping of the interacting regions identified a TDP1/LigIIIα compact dimer of heterodimers in which the two LigIII catalytic cores are positioned in the center, whereas the two TDP1 molecules are located at the edges of the core complex flanked by highly flexible regions that can interact with other repair proteins and SSBs. As TDP1and LigIIIα together repair adducts caused by TOP1 cancer chemotherapy inhibitors, the defined interaction architecture and regulation of this enzyme complex provide insights into a key repair pathway in nonmalignant and cancer cells.     

EAAT4 phosphorylation at the SGK1 consensus site is required for transport modulation by the kinase

EAAT4 (SLC1A6) is a Purkinje-Cell-specific post-synaptic excitatory amino acid transporter that plays a major role in clearing synaptic glutamate. EAAT4 abundance and function is known to be modulated by the serum and glucocorticoid inducible kinase (SGK) 1 but the precise mechanism of kinase action has not been defined yet. The present work aims to identify the molecular mechanism of EAAT4 modulation by the kinase. The EAAT4 sequence bears two putative SGK1 consensus sites (at Thr40 and Thr504) at the amino and carboxy terminus that are conserved among species. Expression studies in Xenopus oocytes demonstrated that EAAT4-mediated [(3)H] glutamate uptake and cell surface abundance are enhanced by co-expression of SGK1. Disruption of the SGK1 phosphorylation site at threonine 40 ((T40A)EAAT4) or of both phosphorylation sites ((T40AT504A)EAAT4) abrogated the effect of SGK1 on transporter function and expression. SGK1 modulates several transport proteins via inhibition of the ubiquitin ligase Nedd4-2. Co-expression of Nedd4-2 inhibited wild-type EAAT4 but not the (T40AT504A)EAAT4 mutant. Besides, RNA interference-mediated reduction of endogenous Nedd4-2 (xNedd4-2) expression increased the activity of the transporter. In conclusion, maximal glutamate transport modulation by SGK1 is accomplished by direct EAAT4 stimulation and to a lesser extent by inhibition of intrinsic Nedd4-2.