Madin-Darby Canine Kidney Epithelial Cells Research Articles

Expression and interaction of two compound heterozygous distal renal tubular acidosis mutants of kidney anion exchanger 1 in epithelial cells

Kidney AE1 (kAE1) is a glycoprotein responsible for the electroneutral exchange of chloride for bicarbonate, promoting the reabsorption of bicarbonate into the blood by alpha-intercalated cells of the collecting tubule. Mutations occurring in the gene encoding kAE1 can induce defects in urinary acidification resulting in distal renal tubular acidosis (dRTA). We expressed two kAE1 dRTA mutants, A858D, a mild dominant mutation, and DeltaV850, a recessive mutation, in epithelial Madin-Darby canine kidney (MDCK) cells. Individuals heterozygous with wild-type (WT) kAE1 either did not display any symptoms of dRTA (DeltaV850/WT) or displayed a mild incomplete form of dRTA (A858D/WT), while compound heterozygotes (DeltaV850/A858D) had dRTA. We found that the A858D mutant was slightly impaired in the endoplasmic reticulum (ER) exit but could target to the basolateral membrane of polarized MDCK cells. Despite an altered binding to an inhibitor affinity resin, anion transport assays showed that the A858D mutant was functional at the cell surface. The DeltaV850 mutant showed altered binding to the affinity resin but was predominantly retained in the ER, resulting in undetectable AE1 expression at the basolateral membrane. When coexpressed in MDCK cells, the WT protein, and to a lesser extent the A858D mutant, enhanced the cell surface expression of the DeltaV850 mutant. The DeltaV850 mutant also affected the cell surface expression of the A858D mutant. Compound heterozygous (A858D/DeltaV850) patients likely possess a decreased amount of functional anion exchangers at the basolateral membrane of their alpha-intercalated cells, resulting in impaired bicarbonate transport into the blood and defective acid transport into the urine.

Regulated Synthesis and Functions of Laminin 5 in Polarized Madin-Darby Canine Kidney Epithelial Cells

Renal tubular epithelial cells synthesize laminin (LN)5 during regeneration of the epithelium after ischemic injury. LN5 is a truncated laminin isoform of particular importance in the epidermis, but it is also constitutively expressed in a number of other epithelia. To investigate the role of LN5 in morphogenesis of a simple renal epithelium, we examined the synthesis and function of LN5 in the spreading, proliferation, wound-edge migration, and apical-basal polarization of Madin-Darby canine kidney (MDCK) cells. MDCK cells synthesize LN5 only when subconfluent, and they degrade the existing LN5 matrix when confluent. Through the use of small-interfering RNA to knockdown the LN5 alpha3 subunit, we were able to demonstrate that LN5 is necessary for cell proliferation and efficient wound-edge migration, but not apical-basal polarization. Surprisingly, suppression of LN5 production caused cells to spread much more extensively than normal on uncoated surfaces, and exogenous keratinocyte LN5 was unable to rescue this phenotype. MDCK cells also synthesized laminin alpha5, a component of LN10, that independent studies suggest may form an assembled basal lamina important for polarization. Overall, our findings indicate that LN5 is likely to play an important role in regulating cell spreading, migration, and proliferation during reconstitution of a continuous epithelium.

Rab10 Regulates Membrane Transport through Early Endosomes of Polarized Madin-Darby Canine Kidney Cells

Rab10, a protein originally isolated from Madin-Darby Canine Kidney (MDCK) epithelial cells, belongs to a family of Rab proteins that includes Rab8 and Rab13. Although both Rab8 and Rab13 have been found to mediate polarized membrane transport, the function of Rab10 in mammalian cells has not yet been established. We have used quantitative confocal microscopy of polarized MDCK cells expressing GFP chimeras of wild-type and mutant forms of Rab10 to analyze the function of Rab10 in polarized cells. These studies demonstrate that Rab10 is specifically associated with the common endosomes of MDCK cells, accessible to endocytic probes internalized from either the apical or basolateral plasma membrane domains. Expression of mutant Rab10 defective for either GTP hydrolysis or GTP binding increased recycling from early compartments on the basolateral endocytic pathway without affecting recycling from later compartments or the apical recycling pathway. These results suggest that Rab10 mediates transport from basolateral sorting endosomes to common endosomes.

Epithelial cell polarity and hypoxia influence heme oxygenase-1 expression by heme in renal epithelial cells

Induction of heme oxygenase-1 (HO-1) in renal tubules occurs as an adaptive and beneficial response in acute renal failure (ARF) following ischemia and nephrotoxins. Using an in vitro model of polarized Madin-Darby canine kidney (MDCK) epithelial cells, we examined apical and basolateral cell surface sensitivity to HO-1 induction by heme. Basolateral exposure to 5 microM hemin (heme chloride) resulted in higher HO-1 induction than did apical exposure. The peak induction of HO-1 by basolateral application of hemin occurred between 12 and 18 h of exposure and was dose dependent. Similar cell surface sensitivity to hemin-induced HO-1 expression was observed using a mouse cortical collecting duct cell line (94D cells). Hepatocyte growth factor (HGF) is known to decrease cell polarity of MDCK cells. Following pretreatment with HGF, apically applied hemin gave greater stimulation of HO-1 expression, whereas HGF alone did not induce HO-1. We also examined the effect of hypoxia on hemin-mediated HO-1 induction. MDCK cells were subjected to hypoxia (1% O(2)) for 24 h to simulate the effects of ischemic ARF. Under hypoxic conditions, both apical as well as basolateral surfaces of MDCK were more sensitive to HO-1 induction by hemin. Hypoxia alone did not induce HO-1 but appeared to potentiate both apical and basolateral sensitivity to hemin-mediated induction. These data demonstrate that the induction of HO-1 expression in polarized renal epithelia by heme is achieved primarily via basolateral exposure. However, under conditions of altered renal epithelial cell polarity and hypoxia, increased HO-1 induction occurs following apical exposure to heme.

Lixazinone Stimulates Mitogenesis of Madin-Darby Canine Kidney Cells

Polycystic kidney diseases (PKD) are characterized by excessive proliferation of renal tubular epithelial cells, development of fluid-filled cysts, and progressive renal insufficiency. cAMP inhibits proliferation of normal renal tubular epithelial cells but stimulates proliferation of renal tubular epithelial cells derived from patients with PKD. Madin-Darby canine kidney (MDCK) epithelial cells, which are widely used as an in vitro model of cystogenesis, also proliferate in response to cAMP. Intracellular cAMP levels are tightly regulated by phosphodiesterases (PDE). Isoform-specific PDE inhibitors have been developed as therapeutic agents to regulate signaling pathways directed by cAMP. In other renal cell types, we have previously demonstrated that cAMP is hydrolyzed by PDE3 and PDE4, but only PDE3 inhibitors suppress proliferation by inhibiting Raf-1 activity (Cheng J, Thompson MA, Walker HJ, Gray CE, Diaz Encarnacion MM, Warner GM, Grande JP. Am J Physiol Renal Physiol 287:F940-F953, 2004.) A potential role for PDE isoform(s) in cAMP-mediated proliferation of MDCK cells has not previously been established. Similar to what we have previously found in several other renal cell types, cAMP hydrolysis in MDCK cells is directed primarily by PDE4 (85% of total activity) and PDE3 (15% of total activity). PDE4 inhibitors are more effective than PDE3 inhibitors in increasing intracellular cAMP levels in MDCK cells. However, only PDE3 inhibitors, and not PDE4 inhibitors, stimulate mitogenesis of MDCK cells. PDE3 but not PDE4 inhibitors activate B-Raf but not Raf-1, as assessed by an in vitro kinase assay. PDE3 but not PDE4 inhibitors activate the ERK pathway and activate cyclins D and E, as assessed by histone H1 kinase assay. We conclude that mitogenesis of MDCK cells is regulated by a functionally compartmentalized intracellular cAMP pool directed by PDE3. Pharmacologic agents that stimulate PDE3 activity may provide the basis for new therapies directed toward reducing cystogenesis in patients with PKD.

Identification and Functional Characterization of Three Chicken Cathelicidins with Potent Antimicrobial Activity

Cathelicidins comprise a family of antimicrobial peptides sharing a highly conserved cathelin domain. Here we report that the entire chicken genome encodes three cathelicidins, namely fowlicidin-1 to -3, which are densely clustered within a 7.5-kb distance at the proximal end of chromosome 2p. Each fowlicidin gene adopts a fourexon, three-intron structure, typical for a mammalian cathelicidin. Phylogenetic analysis revealed that fowlicidins and a group of distantly related mammalian cathelicidins known as neutrophilic granule proteins are likely to originate from a common ancestral gene prior to the separation of birds from mammals, whereas other classic mammalian cathelicidins may have been duplicated from the primordial gene for neutrophilic granule proteins after mammals and birds are diverged. Similar to ovine cathelicidin SMAP-29, putatively mature fowlicidins displayed potent and salt-independent activities against a range of Gram-negative and Gram-positive bacteria, including antibiotic-resistant strains, with minimum inhibitory concentrations in the range of 0.4-2.0 microm for most strains. Fowlicidin-1 and -2 also showed cytotoxicity, with 50% killing of mammalian erythrocytes or epithelial cells in the range of 6-40 microm. In addition, two fowlicidins demonstrated a strong positive cooperativity in binding lipopolysaccharide (LPS), resulting in nearly complete blockage of LPS-mediated proinflammatory gene expression in RAW264.7 cells. Taken together, fowlicidin-1 and -2 are clearly among the most potent cathelicidins that have been reported. Their broad spectrum and salt-insensitive antibacterial activities, coupled with their potent LPS-neutralizing activity, make fowlicidins excellent candidates for novel antimicrobial and anti-sepsis agents.

Zonula Occludens-1 Function in the Assembly of Tight Junctions in Madin-Darby Canine Kidney Epithelial Cells

Zonula occludens (ZO)-1 was the first tight junction protein to be cloned and has been implicated as an important scaffold protein. It contains multiple domains that bind a diverse set of junction proteins. However, the molecular functions of ZO-1 and related proteins such as ZO-2 and ZO-3 have remained unclear. We now show that gene silencing of ZO-1 causes a delay of approximately 3 h in tight junction formation in Madin-Darby canine kidney (MDCK) epithelial cells, but mature junctions seem functionally normal even in the continuing absence of ZO-1. Depletion of ZO-2, cingulin, or occludin, proteins that can interact with ZO-1, had no discernible effects on tight junctions. Rescue of junction assembly using murine ZO-1 mutants demonstrated that the ZO-1 C terminus is neither necessary nor sufficient for normal assembly. Moreover, mutation of the PDZ1 domain did not block rescue. However, point mutations in the Src homology 3 (SH3) domain almost completely prevented rescue. Surprisingly, the isolated SH3 domain of ZO-1 could also rescue junction assembly. These data reveal an unexpected function for the SH3 domain of ZO-1 in regulating tight junction assembly in epithelial cells and show that cingulin, occludin, or ZO-2 are not limiting for junction assembly in MDCK monolayers.

Differences in the apical and basolateral pathways for glycosaminoglycan biosynthesis in Madin–Darby canine kidney cells

Serglycin with a green fluorescent protein tag (SG-GFP) expressed in epithelial Madin-Darby canine kidney cells is secreted mainly (85%) into the apical medium, but the glycosaminoglycan (GAG) chains on the SG-GFP protein core secreted basolaterally (15%) carry most of the sulfate added during biosynthesis (Tveit et al. (2005) J. Biol. Chem., 280, 29596-29603). Here we report further differences in apical and basolateral GAG synthesis. The less intensely sulfated chondroitin sulfate (CS) chains on apically secreted SG-GFP are longer than CS chains attached to basolateral SG-GFP, whereas the heparan sulfate (HS) chains are of similar lengths. When the supply of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) is limited by chlorate treatment, the synthesis machinery maintains sulfation of HS chains on basolateral SG-GFP until it is inhibited at 50 mM chlorate, whereas basolateral CS chains lose sulfate already at 12.5 mM chlorate and become longer. Apically, incorporation of 35S-sulfate into CS is reduced to a lesser extent at higher chlorate concentrations than basolateral CS, although apical CS is less intensely sulfated than basolateral CS in control cells. Similar to what was found for basolateral HS, sulfation of apical HS was not reduced at chlorate concentrations below 50 mM. Also, protein-free, xyloside-based GAG chains secreted basolaterally are more intensely sulfated than their apical counterpart, supporting the view that separate apical and basolateral pathways exist for GAG synthesis and sulfation. Introduction of benzyl beta-d-xyloside (BX) to the GAG synthesis machinery reduces the apical secretion of SG-GFP dramatically and also the modification of SG-GFP by HS.

The mammalian Scribble polarity protein regulates epithelial cell adhesion and migration through E-cadherin

Scribble (Scrib) is a conserved polarity protein required in Drosophila melanogaster for synaptic function, neuroblast differentiation, and epithelial polarization. It is also a tumor suppressor. In rodents, Scrib has been implicated in receptor recycling and planar polarity but not in apical/basal polarity. We now show that knockdown of Scrib disrupts adhesion between Madin–Darby canine kidney epithelial cells. As a consequence, the cells acquire a mesenchymal appearance, migrate more rapidly, and lose directionality. Although tight junction assembly is delayed, confluent monolayers remain polarized. These effects are independent of Rac activation or Scrib binding to βPIX. Rather, Scrib depletion disrupts E-cadherin–mediated cell–cell adhesion. The changes in morphology and migration are phenocopied by E-cadherin knockdown. Adhesion is partially rescued by expression of an E-cadherin–α-catenin fusion protein but not by E-cadherin–green fluorescent protein. These results suggest that Scrib stabilizes the coupling between E-cadherin and the catenins and are consistent with the idea that mammalian Scrib could behave as a tumor suppressor by regulating epithelial cell adhesion and migration.

Trafficking defects of the Southeast Asian ovalocytosis deletion mutant of anion exchanger 1 membrane proteins

Human AE1 (anion exchanger 1) is a membrane glycoprotein found in erythrocytes and as a truncated form (kAE1) in the BLM (basolateral membrane) of a-intercalated cells of the distal nephron, where they carry out electroneutral chloride/bicarbonate exchange. SAO (Southeast Asian ovalocytosis) is a dominant inherited haematological condition arising from deletion of Ala400-Ala408 in AE1, resulting in a misfolded and transport-inactive protein present in the ovalocyte membrane. Heterozygotes with SAO are able to acidify their urine, without symptoms of dRTA (distal renal tubular acidosis) that can be associated with mutations in kAE1. We examined the effect of the SAO deletion on stability and trafficking of AE1 and kAE1 in transfected HEK-293 (human embryonic kidney) cells and kAE1 in MDCK (Madin-Darby canine kidney) epithelial cells. In HEK-293 cells, expression levels and stabilities of SAO proteins were significantly reduced, and no mutant protein was detected at the cell surface. The intracellular retention of AE1 SAO in transfected HEK-293 cells suggests that erythroid-specific factors lacking in HEK-293 cells may be required for cell-surface expression. Although misfolded, SAO proteins could form heterodimers with the normal proteins, as well as homodimers. In MDCK cells, kAE1 was localized to the cell surface or the BLM after polarization, while kAE1 SAO was retained intracellularly. When kAE1 SAO was co-expressed with kAE1 in MDCK cells, kAE1 SAO was largely retained intracellularly; however, it also co-localized with kAE1 at the cell surface. We propose that, in the kidney of heterozygous SAO patients, dimers of kAE1 and heterodimers of kAE1 SAO and kAE1 traffic to the BLM of a-intercalated cells, while homodimers of kAE1 SAO are retained in the endoplasmic reticulum and are rapidly degraded. This results in sufficient cell-surface expression of kAE1 to maintain adequate bicarbonate reabsorption and proton secretion without dRTA.

The Survival Kinase Mirk/dyrk1B Is Activated through Rac1-MKK3 Signaling

The serine/threonine kinase Mirk/dyrk1B is activated in several solid tumors where it mediates cell survival, but the mechanism by which Mirk is activated in tumors is unknown. We now demonstrate that Mirk is activated as a kinase by signaling from Rac1 to the mitogen-activated protein kinase kinase MKK3. Rac is a Ras superfamily GTPase that, when activated, functions downstream of Ras oncoproteins to promote cell survival, transformation, and membrane ruffling. The constitutively active mutant Rac1QL activated Mirk in several cell types through MKK3, which in turn activated Mirk by phosphorylation. Dominant negative Rac1, dominant negative MKK3, and knockdown of MKK3 by RNA interference inhibited the kinase activity of co-expressed Mirk. E-cadherin ligation in confluent Madin-Darby canine kidney (MDCK) epithelial cells is known to transiently activate Rac1. Mirk was activated by endogenous Rac1 following E-cadherin ligation in confluent MDCK epithelial cells, whereas treatment of confluent MDCK cells with an Rac1 inhibitor decreased Mirk activity. Disruption of cadherin ligation by EGTA or prevention of cadherin ligation by maintenance of cells at subconfluent density blocked activation of Mirk. Engagement of cadherin molecules on subconfluent cells by an E-cadherin/Fc chimeric molecule transiently activated both Rac1 and Mirk with a similar time course. Rac activity is up-regulated in many human tumors and mediates survival signals, which enable tumor cells to evade apoptosis. This study characterizes a new anti-apoptotic signaling pathway that connects Rac1 with a novel downstream effector, Mirk kinase, which has recently been demonstrated to mediate survival in human tumors.

Role of 14-3-3γ in FE65-dependent Gene Transactivation Mediated by the Amyloid β-Protein Precursor Cytoplasmic Fragment

The amyloid beta-protein precursor intracellular domain fragment (AICD) is generated from amyloid beta-protein precursor by consecutive cleavages. AICD is thought to activate FE65-dependent gene expression, but the molecular mechanism remains under consideration. We found that dimeric 14-3-3gamma bound both AICD and FE65 simultaneously, and this binding facilitated FE65-dependent gene transactivation by enhancing the association of AICD with FE65. 14-3-3gamma bound to the 667VTPEER672 motif of AICD and, most interestingly, the phosphorylation of AICD at Thr-668 in this motif inhibited the interaction with 14-3-3gamma and blocked gene transactivation. 14-3-3gamma required a sequence between the WW domain and the first phosphotyrosine interaction domain of FE65 for association with FE65. Deletion of this region blocked 14-3-3gamma binding to FE65 and suppressed AICD-mediated FE65-dependent gene transactivation, although the deletion mutant FE65 was still able to bind Tip60, a histone acetyltransferase that forms a complex with FE65 in the nucleus. Taken together, these data demonstrate that 14-3-3gamma facilitates FE65-dependent gene transactivation by forming a complex containing AICD and FE65, and phosphorylation of AICD down-regulates FE65-dependent gene transactivation through the dissociation of 14-3-3gamma and/or FE65 from AICD. Our findings suggest that multiple interactions of AICD with FE65 and 14-3-3gamma modulate FE65-dependent gene transactivation.

EphA2 Phosphorylates the Cytoplasmic Tail of Claudin-4 and Mediates Paracellular Permeability

Eph receptors and ephrin ligands are widely expressed in epithelial cells and mediate cell-cell interaction. EphA2 is expressed in various cancer tissues and cell lines. Although the mechanism of action of EphA2 is unknown, its expression correlates with progression of the malignant phenotype of cancerous tissues. Here, we have shown that EphA2 modulates the localization and function of claudin-4, a constituent of tight junctions. EphA2 associates with claudin-4 via their extracellular domains. This association, in turn, leads to phosphorylation of the cytoplasmic carboxyl terminus of claudin-4 at Tyr-208. The tyrosine phosphorylation of claudin-4 attenuates association of claudin-4 with ZO-1, decreasing integration of claudin-4 into sites of cell-cell contact and enhancing paracellular permeability. These results indicate that EphA2 moderates the function of tight junctions via phosphorylation of claudin-4.

Tumor Suppressor in Lung Cancer (TSLC)1 Suppresses Epithelial Cell Scattering and Tubulogenesis

The tumor suppressor in lung cancer 1 (TSLC1/IGSF4) encodes an immunoglobulin-superfamily cell adhesion molecule whose cytoplasmic domain contains a protein 4.1-binding motif (protein 4.1-BM) and a PDZ-binding motif (PDZ-BM). Loss of TSLC1 expression is frequently observed in advanced cancers implying its involvement in tumor invasion and/or metastasis. Using Madin-Darby canine kidney cells expressing a full-length TSLC1 or various cytoplasmic deletion mutants of TSLC1, we examined the role of TSLC1 in epithelial mesenchymal transitions during the hepatocyte growth factor (HGF)-induced tubulogenesis and cell scattering. In a three-dimensional culture, the full-length TSLC1, which was localized to the lateral membrane of Madin-Darby canine kidney cysts, inhibited HGF-induced tubulogenesis. In contrast, the mutants lacking either the protein 4.1-BM or the PDZ-BM abolished the inhibitory effect on tubulogenesis. In addition, these mutants showed aberrant subcellular localization indicating that lateral localization is correlated with the effect of TSLC1. In a two-dimensional culture, the full-length TSLC1, but not the mutants lacking the protein 4.1-BM or the PDZ-BM, suppressed HGF-induced cell scattering. Furthermore, the cells expressing full-length TSLC1 retained E-cadherin-based cell-cell adhesion even after being treated with HGF. These cells showed prolonged activation of Rac and low activity of Rho, whereas the HGF-treated parental cells induced transient activation of Rac and sustained activation of Rho. Prolonged Rac activation caused by the expression of TSLC1 required its cytoplasmic tail. These findings, taken together, suggest that TSLC1 plays a role in suppressing induction of epithelial mesenchymal transitions by regulating the activation of small Rho GTPases.

Dominant and Recessive Distal Renal Tubular Acidosis Mutations of Kidney Anion Exchanger 1 Induce Distinct Trafficking Defects in MDCK Cells

Distal renal tubular acidosis (dRTA), a kidney disease resulting in defective urinary acidification, can be caused by dominant or recessive mutations in the kidney Cl-/HCO3- anion exchanger (kAE1), a glycoprotein expressed in the basolateral membrane of alpha-intercalated cells. We compared the effect of two dominant (R589H and S613F) and two recessive (S773P and G701D) dRTA point mutations on kAE1 trafficking in Madin-Darby canine kidney (MDCK) epithelial cells. In contrast to wild-type (WT) kAE1 that was localized to the basolateral membrane, the dominant mutants (kAE1 R589H and S613F) were retained in the endoplasmic reticulum (ER) in MDCK cells, with a few cells showing in addition some apical localization. The recessive mutant kAE1 S773P, while misfolded and largely retained in the ER in non-polarized MDCK cells, was targeted to the basolateral membrane after polarization. The other recessive mutants, kAE1 G701D and designed G701E, G701R but not G701A or G701L mutants, were localized to the Golgi in both non-polarized and polarized cells. The results suggest that introduction of a polar mutation into a transmembrane segment resulted in Golgi retention of the recessive G701D mutant. When coexpressed, the dominant mutants retained kAE1 WT intracellularly, while the recessive mutants did not. Coexpression of recessive G701D and S773P mutants in polarized cells showed that these proteins could interact, yet no G701D mutant was detected at the basolateral membrane. Therefore, compound heterozygous patients expressing both recessive mutants (G701D/S773P) likely developed dRTA due to the lack of a functional kAE1 at the basolateral surface of alpha-intercalated cells.

Angiotensin-converting Enzyme 2 (ACE2), But Not ACE, Is Preferentially Localized to the Apical Surface of Polarized Kidney Cells

Angiotensin-converting enzyme-2 (ACE2) is a homologue of angiotensin-I converting enzyme (ACE), the central enzyme of the renin-angiotensin system (RAS). ACE2 is abundant in human kidney and heart and has been implicated in renal and cardiac function through its ability to hydrolyze Angiotensin II. Although ACE2 and ACE are both type I integral membrane proteins and share 61% protein sequence similarity, they display distinct modes of enzyme action and tissue distribution. This study characterized ACE2 at the plasma membrane of non-polarized Chinese hamster ovary (CHO) cells and polarized Madin-Darby canine kidney (MDCKII) epithelial cells and compared its cellular localization to its related enzyme, ACE, using indirect immunofluorescence, cell-surface biotinylation, Western analysis, and enzyme activity assays. This study shows ACE2 and ACE are both cell-surface proteins distributed evenly to detergent-soluble regions of the plasma membrane in CHO cells. However, in polarized MDCKII cells under steady-state conditions the two enzymes are differentially expressed. ACE2 is localized predominantly to the apical surface ( approximately 92%) where it is proteolytically cleaved within its ectodomain to release a soluble form. Comparatively, ACE is present on both the apical ( approximately 55%) and basolateral membranes ( approximately 45%) where it is also secreted but differentially; the ectodomain cleavage of ACE is 2.5-fold greater from the apical surface than the basolateral surface. These studies suggest that both ACE2 and ACE are ectoenzymes that have distinct localization and secretion patterns that determine their role on the cell surface in kidney epithelium and in urine.

Protein Kinase A-dependent Phosphorylation of Lutheran/Basal Cell Adhesion Molecule Glycoprotein Regulates Cell Adhesion to Laminin α5

Lutheran (Lu) blood group and basal cell adhesion molecule (B-CAM) antigens reside on two glycoprotein (gp) isoforms Lu and Lu(v13) that belong to the Ig superfamily and differ only by the size of their cytoplasmic tail. Lu/B-CAM gps have been recognized as laminin alpha5 receptors on red blood cells and epithelial cells in multiple tissues. It has been shown that sickle red cells exhibit enhanced adhesion to laminin alpha5 when intracellular cAMP is up-regulated by physiological stimuli such as epinephrine and that this signaling pathway is protein kinase A- and Lu/B-CAM-dependent. In this study, we analyzed the relationship between the phosphorylation status of Lu/B-CAM gps and their adhesion function to laminin alpha5. We showed that Lu isoform was phosphorylated in sickle red cells as well as in erythroleukemic K562 and epithelial Madin-Darby canine kidney cells and that this phosphorylation is enhanced by different stimuli of the PKA pathway. Lu gp is phosphorylated by glycogen synthase kinase 3 beta, casein kinase II, and PKA at serines 596, 598, and 621, respectively. Alanine substitutions of serines 596 and 598 abolished phosphorylation by glycogen synthase kinase 3 beta and casein kinase II, respectively, but had no effect on adhesion of K562 cells to laminin under flow conditions. Conversely, mutation of serine 621 prevented phosphorylation by PKA and dramatically reduced cell adhesion. Furthermore, stimulation of K562 cells by epinephrine increased Lu gp phosphorylation by PKA and enhanced adhesion to laminin. It is postulated that modulation of the phosphorylation state of Lu gp might be a critical factor for the sickle red cells adhesiveness to laminin alpha5 in sickle cell disease.

Choline Kinase Is a Novel Oncogene that Potentiates RhoA-Induced Carcinogenesis

Choline kinase is overexpressed in human breast, lung, colorectal, and prostate tumors, a finding that suggests the involvement of this enzyme in carcinogenesis. Here we show that overexpression of choline kinase induce oncogenic transformation of human embryo kidney fibroblasts and canine epithelial Madin-Darby canine kidney cells. Choline kinase lays downstream of RhoA signaling and is activated through ROCK kinase, one of the best-characterized RhoA effectors. In keeping with this, coexpression of RhoA and choline kinase potentiates both anchorage independent growth and tumorigenesis. Finally, choline kinase-mediated transformation is sensitive to MN58b, a well-characterized specific choline kinase inhibitor. These results provide the definitive evidence that choline kinase has oncogenic properties and that choline kinase inhibition constitutes a novel valid antitumor strategy.

Small GTPase Proteins Rin and Rit Bind to PAR6 GTP-dependently and Regulate Cell Transformation

The novel small GTPases Rin and Rit are close relatives of Ras, and recent studies show that they play a role in mediating neuronal differentiation. However, the direct effectors of Rin and Rit have yet to be fully characterized. Here we showed that Rin and Rit directly bind to the PDZ domain of PAR6, a cell polarity-regulating protein, in a GTP-dependent manner both in vivo and in vitro. Moreover, Rin and Rit can form a ternary complex consisting of PAR6 and Rac/Cdc42, members of the Rho family of small GTPases modulating cell growth and polarity. This ternary complex synergistically potentiates cell transformation in NIH3T3 cells, and the interaction between Rin/Rit and the PDZ domain of PAR6 is important for this effect. These results suggest that the Rin/Rit-PAR6-Rac/Cdc42 ternary complex may work physiologically in the cells, such as in tumorigenesis.

Absence of N-linked glycosylation does not affect plasma membrane localization of breast cancer resistance protein (BCRP/ABCG2)

Breast cancer resistance protein (BCRP/ABCG2) is an ATP-binding cassette (ABC) multidrug transporter that confers resistance to various anticancer drugs like topotecan and mitoxantrone. To obtain more insight in its cellular functioning, we investigated phosphorylation and N-linked glycosylation of BCRP. In the epithelial Madin-Darby canine kidney (MDCK) cell line, we did not detect phosphorylation of BCRP, in contrast to MRP2, which was phosphorylated. In the ovarian carcinoma cell line T8 also no phosphorylated BCRP was detected. As BCRP in both lines effectively transports drugs, it appears that phosphorylation of BCRP (if it occurs at all) is not needed for drug transport. We further mutated the asparagine residues 418, 557 and 596 in three putative N-linked glycosylation motifs of BCRP to alanines. Mutant proteins were expressed in CHO9 and MDCKII cells by transient transfection and characterized by Western blot and immunofluorescence analysis. We found that only BCRP-N596A and a mutant with all three asparagines mutated (triple mutant) were not glycosylated anymore, indicating that only asparagine 596 is normally glycosylated. The mutation of asparagine 596 (or 418) had little effect on the subcellular localization of BCRP, indicating that N-linked glycosylation is not essential for routing to the plasma membrane. However, BCRP-N557A and the triple mutant were mainly localized intracellularly, probably in the endoplasmic reticulum, suggesting that this mutation disrupted proper routing of BCRP.