CaMKK2 facilitates Golgi-associated vesicle trafficking to sustain cancer cell proliferation

Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) regulates cell and whole-body metabolism and supports tumorigenesis. The cellular impacts of perturbing CAMKK2 expression are, however, not yet fully characterised. By knocking down CAMKK2 levels, we have identified a number of significant subcellular changes indicative of perturbations in vesicle trafficking within the endomembrane compartment. To determine how they might contribute to effects on cell proliferation, we have used proteomics to identify Gemin4 as a direct interactor, capable of binding CAMKK2 and COPI subunits. Prompted by this, we confirmed that CAMKK2 knockdown leads to concomitant and significant reductions in δ-COP protein. Using imaging, we show that CAMKK2 knockdown leads to Golgi expansion, the induction of ER stress, abortive autophagy and impaired lysosomal acidification. All are phenotypes of COPI depletion. Based on our findings, we hypothesise that CAMKK2 sustains cell proliferation in large part through effects on organelle integrity and membrane trafficking.Subject terms: Prostate cancer, Golgi     

A complete map of the Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) signaling pathway

Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) is a serine/threonine-protein kinase belonging to the Ca²⁺/calmodulin-dependent protein kinase subfamily. CAMKK2 has an autocatalytic site, which gets exposed when Ca²⁺/calmodulin (CAM) binds to it. This results in autophosphorylation and complete activation of CAMKK2. The three major known downstream targets of CAMKK2 are 5′-adenosine monophosphate (AMP)-activated protein kinase (AMPKα), calcium/calmodulin-dependent protein kinase 1 (CAMK1) and calcium/calmodulin-dependent protein kinase 4 (CAMK4). Activation of these targets by CAMKK2 is important for the maintenance of different cellular and physiological processes within the cell. CAMKK2 is found to be important in neuronal development, bone remodeling, adipogenesis, and systemic glucose homeostasis, osteoclastgensis and postnatal myogensis. CAMKK2 is reported to be involved in pathologies like Duchenne muscular dystrophy, inflammation, osteoporosis and bone remodeling and is also reported to be overexpressed in prostate cancer, hepatic cancer, ovarian and gastric cancer. CAMKK2 is involved in increased cell proliferation and migration through CAMKK2/AMPK pathway in prostate cancer and activation of AKT in ovarian cancer. Although CAMKK2 is a molecule of great importance, a public resource of the CAMKK2 signaling pathway is currently lacking. Therefore, we carried out detailed data mining and documentation of the signaling events associated with CAMKK2 from published literature and developed an integrated reaction map of CAMKK2 signaling. This resulted in the cataloging of 285 reactions belonging to the CAMKK2 signaling pathway, which includes 33 protein–protein interactions, 74 post-translational modifications, 7 protein translocation events, and 22 activation/inhibition events. Besides, 124 gene regulation events and 25 activator/inhibitors involved in CAMKK2 activation were also cataloged. The CAMKK2 signaling pathway map data is made freely accessible through WikiPathway database (https://www.wikipathways.org/index.php/Pathway:WP4874). We expect that data on a signaling map of CAMKK2 will provide the scientific community with an improved platform to facilitate further molecular as well as biomedical investigations on CAMKK2 and its utility in the development of biomarkers and therapeutic targets.Electronic supplementary materialThe online version of this article (10.1007/s12079-020-00592-1) contains supplementary material, which is available to authorized users.     

Viroporin-mediated calcium-activated autophagy

Autophagy is a cellular response activated by many pathogens, but the mechanism of activation is largely unknown. Recently we showed for the first time that rotavirus initiates the autophagy pathway through a calcium-mediated mechanism. Expression of the rotavirus-encoded NSP4, a pore-forming protein (viroporin), elicits the release of endoplasmic reticulum (ER) lumenal calcium into the cytoplasm of the infected cell. The increased cytoplasmic calcium activates a calcium signaling pathway involving calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) and 5′ adenosine monophosphate-activated protein kinase (AMPK) to trigger autophagy. Rotavirus further manipulates autophagy membrane trafficking to transport viral ER-associated proteins to viroplasms, sites of viral genome replication and immature particle assembly. Transport of viral proteins to viroplasms is required for assembly of infectious virus. Thus, NSP4, a multifunctional viral protein known to regulate infectious particle assembly, also modulates membrane trafficking by orchestrating the activation of autophagy to benefit viral replication.     

Maintaining low Ca2+ level in the endoplasmic reticulum restores abnormal endogenous F508del-CFTR trafficking in airway epithelial cells

The most common mutation in cystic fibrosis, F508del, results in cystic fibrosis transmembrane conductance regulator protein (CFTR) that is retained in the endoplasmic reticulum (ER). Retention is dependent on chaperone proteins, many of which, like calnexin, require calcium for optimal activity. Here, we show that a limited and a maintained ER calcium level is sufficient to inhibit the F508del-CFTR/calnexin interaction and to restore the cAMP-dependent CFTR chloride transport, thus showing the correction of abnormal trafficking. We used Western blot analysis, iodide efflux and calcium measurement techniques applied to the human airway epithelial cystic fibrosis cell line CF15 (F508del/F508del). The inhibition of ER calcium pump, with thapsigargin, curcumin, 2,5-di(t-butyl)hydroquinone or cyclopiazonic acid, maintains a threshold levels of calcium that is correlated to the recovery of endogenous F508del-CFTR transport activity. In particular, cyclopiazonic acid restores a 2-aminoethyoxydiphenyl borate-sensitive F508del-CFTR trafficking with an EC50 of 915 nm. By contrast, the 1,4,5-trisphosphate or IP3 receptor activators, i.e., ATP and histamine, while transiently emptying the ER intracellular calcium store, did not affect the trafficking of F508del-CFTR. Our data suggest that decreasing the ER calcium level is not sufficient to restore the defective trafficking of F508del-CFTR, whereas decreasing and also maintaining low ER calcium level allow correction of defective biosynthetic pathway of endogenous F508del-CFTR in human airway epithelial cells.     

TF — A novel cell-permeable and selective inhibitor of human protein kinase CK2 induces apoptosis in the prostate cancer cell line LNCaP

BackgroundAbnormally high activity of protein kinase CK2 is linked to various diseases including cancer. Therefore, the inhibition of CK2 is a promising therapeutic strategy to fight this disease.MethodsWe screened a library of synthetic molecules concerning their capacity to inhibit CK2. The activity of CK2 and their IC₅₀ and K_i values were determined by a capillary electrophoresis assay. The effects of the inhibitor in a cell culture model were analyzed by cell counting, a viability assay, cytofluorimetry and Western blot.ResultsThe best CK2 inhibitor found in this screen was 6,7-dichloro-1,4-dihydro-8-hydroxy-4-[(4-methylphenylamino)methylen]dibenzo [b,d]furan-3(2H)-one, which we refer to as “TF”. TF showed tight binding to CK2 with low IC₅₀ (29 nM) and K_i (15 nM) values. TF inhibited only seven out of 61 human kinases tested (> 70% inhibition). Incubation of LNCaP cells with 50 μM TF for 48 h decreased the intracellular CK2 activity by 50%, confirming that the inhibitor is membrane permeable. The decrease in activity was correlated with a severe reduction in cell viability. The reduction in cell viability is at least partly due to the induction of apoptosis.General significanceIn many cancers the protein kinase CK2 is significantly up-regulated and supports the neoplastic phenotype. New therapeutic strategies should be based on diverse reliable inhibitors to reverse the abnormal high levels to normal settings.     

Involvement of complex sphingolipids and phosphatidylserine in endosomal trafficking in yeast Saccharomyces cerevisiae

Sphingolipids play critical roles in many physiologically important events in the yeast Saccharomyces cerevisiae. In this study, we found that csg2Δ mutant cells defective in the synthesis of mannosylinositol phosphorylceramide exhibited abnormal intracellular accumulation of an exocytic v‐SNARE, Snc1, under phosphatidylserine synthase gene (PSS1)‐repressive conditions, although in wild‐type cells, Snc1 was known to cycle between plasma membranes and the late Golgi via post‐Golgi endosomes. The mislocalized Snc1 was co‐localized with an endocytic marker dye, FM4‐64, upon labelling for a short time. The abnormal distribution of Snc1 was suppressed by deletion of GYP2 encoding a GTPase‐activating protein that negatively regulates endosomal vesicular trafficking, or expression of GTP‐restricted form of Ypt32 GTPase. Furthermore, an endocytosis‐deficient mutant of Snc1 was localized to plasma membranes in PSS1‐repressed csg2Δ mutant cells as well as wild‐type cells. Thus, the PSS1‐repressed csg2Δ mutant cells were indicated to be defective in the trafficking of Snc1 from post‐Golgi endosomes to the late Golgi. In contrast, the vesicular trafficking pathways via pre‐vacuolar endosomes in the PSS1‐repressed csg2Δ mutant cells seemed to be normal. These results suggested that specific complex sphingolipids and phosphatidylserine are co‐ordinately involved in specific vesicular trafficking pathway.     

An orally available,brain-penetrant CAMKK2 inhibitor reduces food intake in rodent model

Hypothalamic CAMKK2 represents a potential mechanism for chemically affecting satiety and promoting weight loss in clinically obese patients. Single-digit nanomolar inhibitors of CAMKK2 were identified in three related ATP-competitive series. Limited optimization of kinase selectivity, solubility, and pharmacokinetic properties were undertaken on all three series, as SAR was often transferrable. Ultimately, a 2,4-diaryl 7-azaindole was optimized to afford a tool molecule that potently inhibits AMPK phosphorylation in a hypothalamus-derived cell line, is orally bioavailable, and crosses the blood–brain barrier. When dosed orally in rodents, compound 4?t limited ghrelin-induced food intake.     

Interaction of the Deubiquitinating Enzyme Ubp2 and the E3 Ligase Rsp5 Is Required for Transporter/Receptor Sorting in the Multivesicular Body Pathway

Protein ubiquitination is essential for many events linked to intracellular protein trafficking. We sought to elucidate the possible involvement of the S. cerevisiae deubiquitinating enzyme Ubp2 in transporter and receptor trafficking after we (this study) and others established that affinity purified Ubp2 interacts stably with the E3 ubiquitin ligase Rsp5 and the (ubiquitin associated) UBA domain containing protein Rup1. UBP2 interacts genetically with RSP5, while Rup1 facilitates the tethering of Ubp2 to Rsp5 via a PPPSY motif. Using the uracil permease Fur4 as a model reporter system, we establish a role for Ubp2 in membrane protein turnover. Similar to hypomorphic rsp5 alleles, cells deleted for UBP2 exhibited a temporal stabilization of Fur4 at the plasma membrane, indicative of perturbed protein trafficking. This defect was ubiquitin dependent, as a Fur4 N-terminal ubiquitin fusion construct bypassed the block and restored sorting in the mutant. Moreover, the defect was absent in conditions where recycling was absent, implicating Ubp2 in sorting at the multivesicular body. Taken together, our data suggest a previously overlooked role for Ubp2 as a positive regulator of Rsp5-mediated membrane protein trafficking subsequent to endocytosis.     

Store-operated calcium entry-activated autophagy protects EPC proliferation via the CAMKK2-MTOR pathway in ox-LDL exposure

Improving biological functions of endothelial progenitor cells (EPCs) is beneficial to maintaining endothelium homeostasis and promoting vascular re-endothelialization. Because macroautophagy/autophagy has been documented as a double-edged sword in cell functions, its effects on EPCs remain to be elucidated. This study was designed to explore the role and molecular mechanisms of store-operated calcium entry (SOCE)-activated autophagy in proliferation of EPCs under hypercholesterolemia. We employed oxidized low-density lipoprotein (ox-LDL) to mimic hypercholesterolemia in bone marrow-derived EPCs from rat. Ox-LDL dose-dependently activated autophagy flux, while inhibiting EPC proliferation. Importantly, inhibition of autophagy either by silencing Atg7 or by 3-methyladenine treatment, further aggravated proliferative inhibition by ox-LDL, suggesting the protective effects of autophagy against ox-LDL. Interestingly, ox-LDL increased STIM1 expression and intracellular Ca²⁺ concentration. Either Ca²⁺ chelators or deficiency in STIM1 attenuated ox-LDL-induced autophagy activation, confirming the involvement of SOCE in the process. Furthermore, CAMKK2 (calcium/calmodulin-dependent protein kinase kinase 2, β) activation and MTOR (mechanistic target of rapamycin [serine/threonine kinase]) deactivation were associated with autophagy modulation. Together, our results reveal a novel signaling pathway of SOCE-CAMKK2 in the regulation of autophagy and offer new insights into the important roles of autophagy in maintaining proliferation and promoting the survival capability of EPCs. This may be beneficial to improving EPC transplantation efficacy and enhancing vascular re-endothelialization in patients with hypercholesterolemia.     

Polymorphism rs7214723 in CAMKK1: a new genetic variant associated with cardiovascular diseases

Cardiovascular diseases (CVDs) are the leading cause of deaths worldwide. CVDs have a complex etiology due to the several factors underlying its development including environment, lifestyle, and genetics. Given the role of calcium signal transduction in several CVDs, we investigated via PCR-restriction fragment length polymorphism (RFLP) the single nucleotide polymorphism (SNP) rs7214723 within the calcium/calmodulin-dependent kinase kinase 1 (CAMKK1) gene coding for the Ca²⁺/calmodulin-dependent protein kinase kinase I. The variant rs7214723 causes E375G substitution within the kinase domain of CAMKK1. A cross-sectional study was conducted on 300 cardiac patients. RFLP-PCR technique was applied, and statistical analysis was performed to evaluate genotypic and allelic frequencies and to identify an association between SNP and risk of developing specific CVD. Genotype and allele frequencies for rs7214723 were statistically different between cardiopathic and several European reference populations. A logistic regression analysis adjusted for gender, age, diabetes, hypertension, BMI and previous history of malignancy was applied on cardiopathic genotypic data and no association was found between rs7214723 polymorphism and risk of developing specific coronary artery disease (CAD) and aortic stenosis (AS). These results suggest the potential role of rs7214723 in CVD susceptibility as a possible genetic biomarker.     

Iron overload induces apoptosis of osteoblast cells via eliciting ER stress-mediated mitochondrial dysfunction and p-eIF2α/ATF4/CHOP pathway in vitro

Iron is an essential element for crucial biological function; whereas excess iron sedimentation impairs the main functions of tissues or organs. Cumulative researches have shown that the disturbances in iron metabolism, especially iron overload is closely concatenating with bone loss. Nevertheless, the specific process of iron overload-induced apoptosis in osteoblasts has not been thoroughly studied. In this study, our purpose is to elucidate the mechanism of osteoblast apoptosis induced by iron overload via the MC3T3-E1 cell line. Ferric ammonium citrate (FAC) was utilized to simulate iron overload conditions in vitro. These results showed that treatment with FAC dose-dependently induced the apoptosis of MC3T3-E1 cells at 48 h, dysfunction of iron metabolism, and increased intracellular reactive oxygen species (ROS) levels. Following, FAC does-dependently caused the calcium dyshomeostasis, decreased the calcium concentration in endoplasmic reticulum (ER), but increased the crosstalk between ER and mitochondria, and calcium concentration in the mitochondria. Moreover, FAC dose-dependently decreased mitochondrial membrane potential (MMP) and enhanced the expression of apoptosis related proteins (Bax, Cyto-C and C-caspase3). We furthermore revealed that FAC treatment activated the ER-mediated cell apoptosis via p-eIF2α/ATF4/CHOP pathway in MC3T3-E1 osteoblasts cells. In addition, pretreatment with the N-acetylcysteine (NAC) or Tauroursodeoxycholate Sodium (TUDC) attenuated cell apoptosis, ROS levels, mitochondria fragmentation and ER stress-related protein expression, and recovered the protein expression related to iron metabolism. In conclusion, our finding suggested that iron overload induced apoptosis via eliciting ER stress, which resulted in mitochondrial dysfunction and activated p-eIF2α/ATF4/CHOP pathway.     

Effect of hepcidin on intracellular calcium in human osteoblasts

Hepcidin is known to increase intracellular iron through binding to and degrading ferroportin, which is a transmembrane protein that transports iron from the intracellular to the outside. However, it is not clear whether hepcidin has a similar effect on intracellular calcium. Here, we investigated the influence of hepcidin on intracellular calcium in human osteoblasts, with or without high environmental iron concentrations. Our data showed that hepcidin (<100 nmol/L) could increase intracellular calcium, and this effect was more significant when cells were exposed to high environmental iron concentrations. To further explore its underlying mechanisms, we pretreated human osteoblasts with Nimodipine, a L-type calcium channel blocker, and Dantrolene, a ryanodine receptor antagonist to inhibit abnormal calcium release from the sarco-endoplasmic reticulum. These treatments had not resulted in any alteration of intracellular calcium in human osteoblasts. Thus, these findings indicate that the increase of intracellular calcium induced by hepcidin is probably due to calcium release from endoplasmic reticulum, which is triggered by calcium influx.     

Calcium-dependent protein kinase 29 modulates PIN-FORMED polarity and Arabidopsis development via its own phosphorylation code

PIN-FORMED (PIN)-mediated polar auxin transport (PAT) is involved in key developmental processes in plants. Various internal and external cues influence plant development via the modulation of intracellular PIN polarity and, thus, the direction of PAT, but the mechanisms underlying these processes remain largely unknown. PIN proteins harbor a hydrophilic loop (HL) that has important regulatory functions; here, we used the HL as bait in protein pulldown screening for modulators of intracellular PIN trafficking in Arabidopsis thaliana. Calcium-dependent protein kinase 29 (CPK29), a Ca²⁺-dependent protein kinase, was identified and shown to phosphorylate specific target residues on the PIN-HL that were not phosphorylated by other kinases. Furthermore, loss of CPK29 or mutations of the phospho-target residues in PIN-HLs significantly compromised intracellular PIN trafficking and polarity, causing defects in PIN-mediated auxin redistribution and biological processes such as lateral root formation, root twisting, hypocotyl gravitropism, phyllotaxis, and reproductive development. These findings indicate that CPK29 directly interprets Ca²⁺ signals from internal and external triggers, resulting in the modulation of PIN trafficking and auxin responses.

Ca²⁺-dependent protein kinase 29 directly phosphorylates the hydrophilic loop of PIN-FORMED proteins to modulate their intracellular trafficking and Arabidopsis development.     

Thyroid hormone induction of mitochondrial activity is coupled to mitophagy via ROS-AMPK-ULK1 signaling

Currently, there is limited understanding about hormonal regulation of mitochondrial turnover. Thyroid hormone (T₃) increases oxidative phosphorylation (OXPHOS), which generates reactive oxygen species (ROS) that damage mitochondria. However, the mechanism for maintenance of mitochondrial activity and quality control by this hormone is not known. Here, we used both in vitro and in vivo hepatic cell models to demonstrate that induction of mitophagy by T₃ is coupled to oxidative phosphorylation and ROS production. We show that T₃ induction of ROS activates CAMKK2 (calcium/calmodulin-dependent protein kinase kinase 2, β) mediated phosphorylation of PRKAA1/AMPK (5′ AMP-activated protein kinase), which in turn phosphorylates ULK1 (unc-51 like autophagy activating kinase 1) leading to its mitochondrial recruitment and initiation of mitophagy. Furthermore, loss of ULK1 in T₃-treated cells impairs both mitophagy as well as OXPHOS without affecting T₃ induced general autophagy/lipophagy. These findings demonstrate a novel ROS-AMPK-ULK1 mechanism that couples T₃-induced mitochondrial turnover with activity, wherein mitophagy is necessary not only for removing damaged mitochondria but also for sustaining efficient OXPHOS.     

Differential Roles of Tubby Family Proteins in Ciliary Formation and Trafficking

Cilia are highly specialized organelles that extend from the cell membrane and function as cellular signaling hubs. Thus, cilia formation and the trafficking of signaling molecules into cilia are essential cellular processes. TULP3 and Tubby (TUB) are members of the tubby-like protein (TULP) family that regulate the ciliary trafficking of G-protein coupled receptors, but the functions of the remaining TULPs (i.e., TULP1 and TULP2) remain unclear. Herein, we explore whether these four structurally similar TULPs share a molecular function in ciliary protein trafficking. We found that TULP3 and TUB, but not TULP1 or TULP2, can rescue the defective cilia formation observed in TULP3-knockout (KO) hTERT RPE-1 cells. TULP3 and TUB also fully rescue the defective ciliary localization of ARL13B, INPP5E, and GPR161 in TULP3 KO RPE-1 cells, while TULP1 and TULP2 only mediate partial rescues. Furthermore, loss of TULP3 results in abnormal IFT140 localization, which can be fully rescued by TUB and partially rescued by TULP1 and TULP2. TUB’s capacity for binding IFT-A is essential for its role in cilia formation and ciliary protein trafficking in RPE-1 cells, whereas its capacity for PIP₂ binding is required for proper cilia length and IFT140 localization. Finally, chimeric TULP1 containing the IFT-A binding domain of TULP3 fully rescues ciliary protein trafficking, but not cilia formation. Together, these two TULP domains play distinct roles in ciliary protein trafficking but are insufficient for cilia formation in RPE-1 cells. In addition, TULP1 and TULP2 play other unknown molecular roles that should be addressed in the future.     

SORTING NEXIN1 Is Required for Modulating the Trafficking and Stability of the Arabidopsis IRON-REGULATED TRANSPORTER1

Dicotyledonous plants growing under limited iron availability initiate a response resulting in the solubilization, reduction, and uptake of soil iron. The protein factors responsible for these steps are transmembrane proteins, suggesting that the intracellular trafficking machinery may be involved in iron acquisition. In search for components involved in the regulation of Arabidopsis thaliana iron deficiency responses, we identified the members of the SORTING NEXIN (SNX) protein family. SNX loss-of-function plants display enhanced susceptibility to iron deficiency in comparison to the wild type. The absence of SNX led to reduced iron import efficiency into the root. SNX1 showed partial colocalization with the principal root iron importer IRON-REGULATED TRANSPORTER1 (IRT1). In SNX loss-of-function plants, IRT1 protein levels were decreased compared with the wild type due to enhanced IRT1 degradation. This resulted in diminished amounts of the IRT1 protein at the plasma membrane. snx mutants exhibited enhanced iron deficiency responses compared with the wild type, presumably due to the lower iron uptake through IRT1. Our results reveal a role of SNX1 for the correct trafficking of IRT1 and, thus, for modulating the activity of the iron uptake machinery.     

A new double-stranded RNA-binding protein that interacts with PKR

We have identified a 74 kDa double-stranded (ds)RNA-binding protein that shares extensive homology with the mouse spermatid perinuclear RNA-binding (Spnr) protein. p74 contains two dsRNA-binding motifs (dsRBMs) that are essential for preferential binding to dsRNA. Previously, dsRNA-binding proteins were shown to undergo homo- and heterodimerization, raising the possibility that regulation of activity could be controlled by interactions between different family members. Homodimerization is required to activate the dsRNA-dependent protein kinase PKR, whereas heterodimerization between PKR and other dsRNA-binding proteins can inhibit kinase activity. We have found that p74 also interacts with PKR, both the wild-type enzyme and a catalytically defective mutant (K296R). While co-expression of p74 and wild-type PKR in the yeast Saccharomyces cerevisiae did not alter PKR activity, co-expression of p74 and the catalytically defective K296R mutant surprisingly resulted in abnormal morphology and cell death in transformants that maintained a high level of p74 expression. These transformants could be rescued by overexpression of the α-subunit of wild-type eukaryotic translation initiation factor 2 (eIF2α), one of the known substrates for PKR. We hypothesize that competing heterodimers between p74–K296R PKR and eIF2α–K296R PKR may control cell growth such that stabilization of the p74–K296R PKR heterodimer induces abnormal morphology and cell death.     

Vasopressin regulated trafficking of a green fluorescent protein-aquaporin 2 chimera in LLC-PK1 cells

 Aquaporin 2 (AQP2) transfected into LLC-PK₁ cells functions as a vasopressin-regulated water channel that recycles between intracellular vesicles and the plasma membrane upon vasopressin stimulation. The green fluorescent protein (GFP) of the jellyfish, Aequorea victoria, was used as an autofluorescent tag to monitor AQP2 trafficking in transfected LLC-PK₁ cells. Two chimeras were constructed, one in which GFP was fused to the amino-terminus of AQP2 [GFP-AQP2(NT)] and the second in which it was fused to the carboxyl-terminus [AQP2-GFP(CT)]. The GFP-AQP2(NT) chimera trafficked in a regulated pathway from intracellular vesicles to the basolateral plasma membrane in response to vasopressin or forskolin stimulation of cells. In contrast, the AQP2-GFP(CT) chimera expressed in LLC-PK₁ cells was localized constitutively on both apical and basolateral plasma membranes. The cellular location of this chimera was not modified by vasopressin or forskolin. Thus, while the GFP-AQP2(NT) chimera will be useful to study AQP2 trafficking in vitro, the abnormal, constitutive membrane localization of the AQP2-GFP(CT) chimera suggests that one or more trafficking signals exist on the carboxyl-terminus of the AQP2 protein. Accepted: 8 April 1998     

Improvement in Lipid and Protein Trafficking in Niemann‐Pick C1 Cells by Correction of a Secondary Enzyme Defect

Different primary lysosomal trafficking defects lead to common alterations in lipid trafficking, suggesting cooperative interactions among lysosomal lipids. However, cellular analysis of the functional consequences of this phenomenon is lacking. As a test case, we studied cells with defective Niemann‐Pick C1 (NPC1) protein, a cholesterol trafficking protein whose defect gives rise to lysosomal accumulation of cholesterol and other lipids, leading to NPC disease. NPC1 cells also develop a secondary defect in acid sphingomyelinase (SMase) activity despite a normal acid SMase gene (SMPD1). When acid SMase activity was restored to normal levels in NPC1‐deficient CHO cells through SMPD1 transfection, there was a dramatic reduction in lysosomal cholesterol. Two other defects, excess lysosomal bis‐(monoacylglycerol) phosphate (BMP) and defective transferrin receptor (TfR) recycling, were also markedly improved. To test its relevance in human cells, the acid SMase activity defect in fibroblasts from NPC1 patients was corrected by SMPD1 transfection or acid SMase enzyme replacement. Both treatments resulted in a dramatic reduction in lysosomal cholesterol. These data show that correcting one aspect of a complex lysosomal lipid storage disease can reduce the cellular consequences even if the primary genetic defect is not corrected.     

A calcium-dependent but calmodulin-independent protein kinase from soybean

A calcium-dependent protein kinase activity from suspension-cultured soybean cells (Glycine max L. Wayne) was shown to be dependent on calcium but not calmodulin. The concentrations of free calcium required for half-maximal histone H1 phosphorylation and autophosphorylation were similar (≈2 micromolar). The protein kinase activity was stimulated 100-fold by ≥10 micromolar-free calcium. When exogenous soybean or bovine brain calmodulin was added in high concentration (1 micromolar) to the purified kinase, calcium-dependent and -independent activities were weakly stimulated (≤2-fold). Bovine serum albumin had a similar effect on both activities. The kinase was separated from a small amount of contaminating calmodulin by sodium dodecyl sulfate polyacrylamide gel electrophoresis. After renaturation the protein kinase autophosphorylated and phosphorylated histone H1 in a calcium-dependent manner. Following electroblotting onto nitrocellulose, the kinase bound ⁴⁵Ca²⁺ in the presence of KCl and MgCl₂, which indicates that the kinase itself is a high-affinity calcium-binding protein. Also, the mobility of one of two kinase bands in SDS gels was dependent on the presence of calcium. Autophosphorylation of the calmodulin-free kinase was inhibited by the calmodulin-binding compound N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7), showing that the inhibition of activity by W-7 is independent of calmodulin. These results show that soybean calcium-dependent protein kinase represents a new class of protein kinase which requires calcium but not calmodulin for activity.