Diacylglycerol kinase α controls RCP-dependent integrin trafficking to promote invasive migration

Inhibition of αvβ3 integrin or expression of oncogenic mutants of p53 promote invasive cell migration by enhancing endosomal recycling of α5β1 integrin under control of the Rab11 effector Rab-coupling protein (RCP). In this paper, we show that diacylglycerol kinase α (DGK-α), which phosphorylates diacylglycerol to phosphatidic acid (PA), was required for RCP to be mobilized to and tethered at the tips of invasive pseudopods and to allow RCP-dependent α5β1 recycling and the resulting invasiveness of tumor cells. Expression of a constitutive-active mutant of DGK-α drove RCP-dependent invasion in the absence of mutant p53 expression or αvβ3 inhibition, and conversely, an RCP mutant lacking the PA-binding C2 domain was not capable of being tethered at pseudopod tips. These data demonstrate that generation of PA downstream of DGK-α is essential to connect expression of mutant p53s or inhibition of αvβ3 to RCP and for this Rab11 effector to drive the trafficking of α5β1 that is required for tumor cell invasion through three-dimensional matrices.     

RCP-driven α5β1 recycling suppresses Rac and promotes RhoA activity via the RacGAP1–IQGAP1 complex

Inhibition of αvβ3 or expression of mutant p53 promotes invasion into fibronectin (FN)-containing extracellular matrix (ECM) by enhancing Rab-coupling protein (RCP)–dependent recycling of α5β1 integrin. RCP and α5β1 cooperatively recruit receptor tyrosine kinases, including EGFR1, to regulate their trafficking and downstream signaling via protein kinase B (PKB)/Akt, which, in turn, promotes invasive migration. In this paper, we identify a novel PKB/Akt substrate, RacGAP1, which is phosphorylated as a consequence of RCP-dependent α5β1 trafficking. Phosphorylation of RacGAP1 promotes its recruitment to IQGAP1 at the tips of invasive pseudopods, and RacGAP1 then locally suppresses the activity of the cytoskeletal regulator Rac and promotes the activity of RhoA in this subcellular region. This Rac to RhoA switch promotes the extension of pseudopodial processes and invasive migration into FN-containing matrices, in a RhoA-dependent manner. Thus, the localized endocytic trafficking of α5β1 within the tips of invasive pseudopods elicits signals that promote the reorganization of the actin cytoskeleton, protrusion, and invasion into FN-rich ECM.     

IP3 sensitizes TRPV4 channel to the mechano- and osmotransducing messenger 5'-6'-epoxyeicosatrienoic acid

Here we show that blocking the adhesive function of αvβ3 integrin with soluble RGD ligands, such as osteopontin or cilengitide, promoted association of Rab-coupling protein (RCP) with α5β1 integrin and drove RCP-dependent recycling of α5β1 to the plasma membrane and its mobilization to dynamic ruffling protrusions at the cell front. These RCP-driven changes in α5β1 trafficking led to acquisition of rapid/random movement on two-dimensional substrates and to a marked increase in fibronectin-dependent migration of tumor cells into three-dimensional matrices. Recycling of α5β1 integrin did not affect its regulation or ability to form adhesive bonds with substrate fibronectin. Instead, α5β1 controlled the association of EGFR1 with RCP to promote the coordinate recycling of these two receptors. This modified signaling downstream of EGFR1 to increase its autophosphorylation and activation of the proinvasive kinase PKB/Akt. We conclude that RCP provides a scaffold that promotes the physical association and coordinate trafficking of α5β1 and EGFR1 and that this drives migration of tumor cells into three-dimensional matrices.     

Analysis of Occludin Trafficking,Demonstrating Continuous Endocytosis,Degradation, Recycling and Biosynthetic Secretory Trafficking

Tight junctions (TJs) link adjacent cells and are critical for maintenance of apical-basolateral polarity in epithelial monolayers. The TJ protein occludin functions in disparate processes, including wound healing and Hepatitis C Virus infection. Little is known about steady-state occludin trafficking into and out of the plasma membrane. Therefore, we determined the mechanisms responsible for occludin turnover in confluent Madin-Darby canine kidney (MDCK) epithelial monolayers. Using various biotin-based trafficking assays we observed continuous and rapid endocytosis of plasma membrane localised occludin (the majority internalised within 30 minutes). By 120 minutes a significant reduction in internalised occludin was observed. Inhibition of lysosomal function attenuated the reduction in occludin signal post-endocytosis and promoted co-localisation with the late endocytic system. Using a similar method we demonstrated that ∼20% of internalised occludin was transported back to the cell surface. Consistent with these findings, significant co-localisation between internalised occludin and recycling endosomal compartments was observed. We then quantified the extent to which occludin synthesis and transport to the plasma membrane contributes to plasma membrane occludin homeostasis, identifying inhibition of protein synthesis led to decreased plasma membrane localised occludin. Significant co-localisation between occludin and the biosynthetic secretory pathway was demonstrated. Thus, under steady-state conditions occludin undergoes turnover via a continuous cycle of endocytosis, recycling and degradation, with degradation compensated for by biosynthetic exocytic trafficking. We developed a mathematical model to describe the endocytosis, recycling and degradation of occludin, utilising experimental data to provide quantitative estimates for the rates of these processes.     

Rab13 regulates membrane trafficking between TGN and recycling endosomes in polarized epithelial cells

To maintain polarity, epithelial cells continuously sort transmembrane proteins to the apical or basolateral membrane domains during biosynthetic delivery or after internalization. During biosynthetic delivery, some cargo proteins move from the trans-Golgi network (TGN) into recycling endosomes (RE) before being delivered to the plasma membrane. However, proteins that regulate this transport step remained elusive. In this study, we show that Rab13 partially colocalizes with TGN38 at the TGN and transferrin receptors in RE. Knockdown of Rab13 with short hairpin RNA in human bronchial epithelial cells or overexpression of dominant-active or dominant-negative alleles of Rab13 in Madin-Darby canine kidney cells disrupts TGN38/46 localization at the TGN. Moreover, overexpression of Rab13 mutant alleles inhibits surface arrival of proteins that move through RE during biosynthetic delivery (vesicular stomatitis virus glycoprotein [VSVG], A-VSVG, and LDLR-CT27). Importantly, proteins using a direct route from the TGN to the plasma membrane are not affected. Thus, Rab13 appears to regulate membrane trafficking between TGN and RE.     

Serine phosphorylation site of the 46-kDa mannose 6-phosphate receptor is required for transport to the plasma membrane in Madin-Darby canine kidney and mouse fibroblast cells.

Up to 4% of the human 46-kDa mannose 6-phosphate receptor (MPR46) expressed in Madin-Darby canine kidney (MDCK) cells are localized at the cell surface. At steady state, the expression of MPR46 on the apical surface of filter-grown MDCK cells is about sixfold lower than on the basolateral surface. The cytoplasmic domain of the MPR46 is phosphorylated on serine 56 at low stoichiometry. By expressing mutant MPR46 we have shown that the MPR46 phosphorylation site is required for delivery to the plasma membrane. In addition, mutant MPR46 expressed in MPR-deficient mouse embryonic fibroblasts were not detected at the cell surface and their ability to sort newly synthesized cathepsin D was not altered. Since the loss of MPR46 phosphorylation correlates with the lack of cell surface expression, phosphorylation of serine 56 may either function as a direct plasma membrane targeting signal or inhibit MPR46 recycling from endosomes to Golgi, resulting in trafficking to the cell surface.     

Mutant p53 exhibits trivial effects on mitochondrial functions which can be reactivated by ellipticine in lymphoma cells

Increasing evidence has shown that a fraction of the wild-type (wt) form of the tumor suppressor p53, can translocate to mitochondria due to genotoxic stress. The mitochondrial targets of wt p53 have also been studied. However, whether mutant p53, which exists in 50% of human cancers, translocates to mitochondria and affects mitochondrial functions is unclear. In this study, we used doxorubicin, a chemotherapeutic drug, to treat five human lymphoma cell lines with wt, mutant or deficient in p53, to induce p53 activation and mitochondrial translocation. Our results demonstrated that mutant p53, like wt p53, was induced upon doxorubicin treatment. Similarly, a fraction of mutant p53 also translocated to mitochondria. However, Complex I and II activities in the mitochondria were compromised only in wt p53-bearing cells after doxorubicin treatment, but not in mutant p53-bearing cells. Similarly, doxorubicin treatment caused greater cell death only in wt p53-bearing cells, but not in mutant p53-bearing cells. When p53 deficient Ramos cells were transfected with mutant p53 (249S), the cells showed resistance to doxorubicin-induced cell death and decreases in complex activities. To reactivate mutant p53 and reverse chemoresistance, ellipticine (5,11-dimethyl-6H-pyrido[4,3-b]carbazole) was used to treat mutant p53 cells. Ellipticine enhanced p53 mitochondrial translocation, decreased Complex I activity, and sensitized p53 mutant cells to doxorubicin-induced apoptosis. In summary, our studies suggest that mutations in p53 may not hinder p53’s mitochondrial translocation, but impair its effects on mitochondrial functions. Therefore, restoring mutant p53 by ellipticine may sensitize these cells to chemotherapy.     

Regulation of vesicle trafficking in madin-darby canine kidney cells by Rab11a and Rab25

Polarized epithelial cells maintain the polarized distribution of basolateral and apical membrane proteins through a process of receptor-mediated endocytosis, sorting, and then recycling to the appropriate membrane domain. We have previously shown that the small GTP-binding proteins, Rab11a and Rab25, are associated with the apical recycling system of Madin-Darby canine kidney cells. Here we have utilized inducible expression of wild-type, dominant negative, and constitutively active mutants to directly compare the functions of Rab25 and Rab11a in postendocytic vesicular transport. We found that a Rab11a mutant deficient in GTP binding, Rab11aS25N, potently inhibited both transcytosis and apical recycling yet failed to inhibit transferrin recycling. Similarly, expression of either wild type Rab25 or the active mutant Rab25S21V inhibited both apical recycling and transcytosis of IgA by greater than 50% but had no effect on basolateral recycling of transferrin. Interestingly, the GTPase-deficient mutant Rab11aS20V inhibited basolateral to apical transcytosis of IgA, but had no effect on either apical or basolateral recycling. These results indicate that neither Rab11a nor Rab25 function in the basolateral recycling of transferrin in polarized Madin-Darby canine kidney cells cells, consistent with recent morphological observations by others. Thus, transferrin receptors must be recycled to the plasma membrane prior to sorting of apically directed cargoes into Rab11a/Rab25-positive apical recycling endosomes.     

Cooperation among Numb, MDM2 and p53 in the development and progression of pancreatic cancer

We study the expression of Numb, MDM2 and p53 for clinical significance in pancreatic cancer (PC) and their functional relationship in regulating biological behaviors of PC cells. IHC, IB and qRT-PCR were used to detect Numb, MDM2 and p53 expression in PC. Transfection and drug intervention were used to investigate their functional relationship in PC cells. IHC showed that Numb expression was negatively associated with tumor size, differentiation and UICC stage, while expression of MDM2 and p53 was positively associated with tumor T and UICC stages, respectively (P?<?0.05). Numb was an independent prognostic indicator in PC (P?<?0.05). Patients with Numb-positive expression or combined with MDM2-negative expression had a significantly better overall survival (P?<?0.05). Altered expression of Numb can regulate wild-type but not mutant p53 expression, while MDM2 knockdown increased Numb but not mutant p53 protein level. Meanwhile, Numb knockdown increased chemoresistance but decreased activated p53 and cleaved-caspase-3 protein expression in gemcitabine-treated Capan-2 cells. Moreover, Numb co-immunoprecipitated with p53 to prevent p53 ubiquitin-dependent protein degradation and this ubiquitin-dependent regulation plays an important role in the coordinate function of these three proteins on cell invasion and migration in PC cells. Our study is the first to demonstrate the clinical significance and functional cooperation among Numb, MDM2 and p53 involved in the development and progression of PC.     

Intrinsic Signals in the Unique Domain Target p56lckto the Plasma Membrane Independently of CD4

In T lymphocytes, the Src-family protein tyrosine kinase p56^lck (Lck) is mostly associated with the cytoplasmic face of the plasma membrane. To determine how this distribution is achieved, we analyzed the location of Lck in lymphoid and in transfected nonlymphoid cells by immunofluorescence. We found that in T cells Lck was targeted correctly, independently of the cell surface proteins CD4 and CD8 with which it interacts. Similarly, in transfected NIH-3T3 fibroblasts, Lck was localized at the plasma membrane, indicating that T cell–specific proteins are not required for targeting. Some variation in subcellular distribution was observed when Lck was expressed in HeLa and MDCK cells. In these cells, Lck associated with both the plasma membrane and the Golgi apparatus, while subsequent expression of CD4 resulted in the loss of Golgi-associated staining. Together, these data indicate that Lck contains intrinsic signals for targeting to the plasma membrane. Furthermore, delivery to this site may be achieved via association with exocytic transport vesicles.

A mutant Lck molecule in which the palmitoylation site at cysteine 5 was changed to lysine (LC2) localized to the plasma membrane and the Golgi region in NIH3T3 cells. However, the localization of a mutant in which the palmitoylation site at cysteine 3 was changed to serine (LC1) was indistinguishable from wild-type Lck. Chimeras composed of only the unique domain of Lck linked to either c-Src or the green fluorescent protein similarly localized to the plasma membrane of NIH-3T3 cells. Thus, the targeting of Lck appears to be determined primarily by its unique domain and may be influenced by the use of different palmitoylation sites.

     

Ordering of compartments in the yeast endocytic pathway

We have characterized the morphology of the yeast endocytic pathway leading from the plasma membrane to the vacuole by following the trafficking of positively charged nanogold in combination with compartment identification using immunolocalization of t-SNARE proteins. The first endocytic compartment, termed the early/recycling endosome, contains the t-SNARE, Tlg1p. The next compartment, the prevacuolar compartment, contains Pep12p. After transport to the prevacuolar compartment, where vacuolar enzymes are seen on their way to the vacuole, endocytic content is delivered to the late endosome and on to the vacuole, both of which are devoid of Pep12p immunolabel. Traffic to the prevacuolar compartment is reduced in strains mutant for the Rab5 homologs, Vps21p, Ypt52p, and Ypt53p and in vps27 mutant cells. On the other hand, traffic to the early recycling endosome is less dependent on Rab5 homologs and does not require Vps27p.     

Expression of wild-type and mutant p53 proteins by recombinant vaccinia viruses.

To facilitate the purification of wild type p53 protein, we established a recombinant p53 vaccinia viral expression system. Using this efficient eukaryotic expression vector, we found that the expressed p53 proteins retained their specific structural characteristics. A comparison between wild type and mutant p53 proteins showed the conservation of the typical subcellular localization and the expression of specific antigenic determinants. Furthermore, wild type p53 exhibited a typical binding with large T antigen, whereas no binding was detected with mutant p53. Both wild type and mutant p53 proteins were highly stable and constituted 5-7% of total protein expressed in the infected cells. These expression recombinant viruses offer a simple, valuable system for the purification of wild type and mutant p53 proteins that are expressed abundantly in eukaryotic cells.     

Copper-dependent degradation of the Saccharomyces cerevisiae plasma membrane copper transporter Ctr1p in the apparent absence of endocytosis.

The cell surface protein repertoire needs to be regulated in response to changes in the extracellular environment. In this study, we investigate protein turnover of the Saccharomyces cerevisiae plasma membrane copper transporter Ctr1p, in response to a change in extra-cellular copper levels. As Ctr1p mediates high affinity uptake of copper into the cell, modulation of its expression is expected to be involved in copper homeostasis. We demonstrate that Ctr1p is a stable protein when cells are grown in low concentrations of copper, but that exposure of cells to high concentrations of copper (10 microM) triggers degradation of cell surface Ctr1p. This degradation appears to be specific for Ctr1p and does not occur with another yeast plasma membrane protein tested. Internalization of some Ctr1p can be seen when cells are exposed to copper. However, yeast mutant strains defective in endocytosis (end3, end4 and chc1-ts) and vacuolar degradation (pep4) exhibit copper-dependent Ctr1p degradation, indicating that internalization and delivery to the vacuole is not the principal mechanism responsible for degradation. In addition, a variant Ctr1p with a deletion in the cytosolic tail is not internalized upon exposure of cells to copper, but is nevertheless degraded. These observations indicate that proteolysis at the plasma membrane most likely explains copper-dependent turnover of Ctr1p and point to the existence of a novel pathway in yeast for plasma membrane protein turnover.     

Sorting nexin 27 regulates basal and stimulated brush border trafficking of NHE3

Sorting nexin 27 (SNX27) contains a PDZ domain that is phylogenetically related to the PDZ domains of the NHERF proteins. Studies on nonepithelial cells have shown that this protein is located in endosomes, where it regulates trafficking of cargo proteins in a PDZ domain–dependent manner. However, the role of SNX27 in trafficking of cargo proteins in epithelial cells has not been adequately explored. Here we show that SNX27 directly interacts with NHE3 (C-terminus) primarily through the SNX27 PDZ domain. A combination of knockdown and reconstitution experiments with wild type and a PDZ domain mutant (GYGF → GAGA) of SNX27 demonstrate that the PDZ domain of SNX27 is required to maintain basal NHE3 activity and surface expression of NHE3 in polarized epithelial cells. Biotinylation-based recycling and degradation studies in intestinal epithelial cells show that SNX27 is required for the exocytosis (not endocytosis) of NHE3 from early endosome to plasma membrane. SNX27 is also required to regulate the retention of NHE3 on the plasma membrane. The findings of the present study extend our understanding of PDZ-mediated recycling of cargo proteins from endosome to plasma membrane in epithelial cells.     

Galectin-1 confers resistance to doxorubicin in hepatocellular carcinoma cells through modulation of P-glycoprotein expression

Galectin-1 (GAL1), a β-galactoside-binding protein abundantly expressed in the tumor microenvironment, has emerged as a key mechanism of chemoresistance developed by different tumors. Although increased expression of GAL1 is a hallmark of hepatocellular carcinoma (HCC) progression, aggressiveness and metastasis, limited information is available on the role of this endogenous lectin in HCC resistance to chemotherapy. Moreover, the precise mechanisms underlying this effect are uncertain. HCC has evolved different mechanisms of resistance to chemotherapy including those involving the P-glycoprotein (P-gp), an ATP-dependent drug efflux pump, which controls intracellular drug concentration. Here, we investigated the molecular mechanism underlying GAL1-mediated chemoresistance in HCC cells, particularly the involvement of P-gp in this effect. Our results show that GAL1 protected HepG2 cells from doxorubicin (DOX)- and sorafenib-induced cell death in vitro. Accordingly, GAL1-overexpressing HepG2 cells generated DOX-resistant tumors in vivo. High expression of GAL1 in HepG2 cells reduced intracellular accumulation of DOX likely by increasing P-gp protein expression rather than altering its membrane localization. GAL1-mediated increase of P-gp expression involved activation of the phosphatidylinositol-3 kinase (PI3K) signaling pathway. Moreover, ‘loss-of-function’ experiments revealed that P-gp mediates GAL1-driven resistance to DOX, but not to sorafenib, in HepG2 cells. Conversely, in PLC/PRF/5 cells, P-gp protein expression was undetectable and GAL1 did not control resistance to DOX or sorafenib, supporting the critical role of P-gp in mediating GAL1 effects. Collectively, our findings suggest that GAL1 confers chemoresistance in HCC through mechanisms involving modulation of P-gp, thus emphasizing the role of this lectin as a potential therapeutic target in HCC.Subject terms: Glycobiology, Liver cancer