Conserved in VivoPhosphorylation of Calnexin at Casein Kinase II Sites as Well as a Protein Kinase C/Proline-directed Kinase Site

Calnexin is a lectin-like chaperone of the endoplasmic reticulum (ER) that couples temporally and spatially N-linked oligosaccharide modifications with the productive folding of newly synthesized glycoproteins. Calnexin was originally identified as a major type I integral membrane protein substrate of kinase(s) associated with the ER. Casein kinase II (CK2) was subsequently identified as an ER-associated kinase responsible for the in vitro phosphorylation of calnexin in microsomes (Ou, W-J., Thomas, D. Y., Bell, A. W., and Bergeron, J. J. M. (1992) J. Biol. Chem. 267, 23789-23796). We now report on the in vivo sites of calnexin phosphorylation. After 32PO4 labeling of HepG2 and Madin-Darby canine kidney cells, immunoprecipitated calnexin was phosphorylated exclusively on serine residues. Using nonradiolabeled cells, we subjected calnexin immunoprecipitates to in gel tryptic digestion followed by nanoelectrospray mass spectrometry employing selective scans specific for detection of phosphorylated fragments. Mass analyses identified three phosphorylated sites in calnexin from either HepG2 or Madin-Darby canine kidney cells. The three sites were localized to the more carboxyl-terminal half of the cytosolic domain: S534DAE (CK2 motif), S544QEE (CK2 motif), and S563PR. We conclude that CK2 is a kinase that phosphorylates calnexin in vivo as well as in microsomes in vitro. Another yet to be identified kinase (protein kinase C and/or proline-directed kinase) is directed toward the most COOH-terminal serine residue. Elucidation of the signaling cascade responsible for calnexin phosphorylation at these sites in vivo may define a novel regulatory function for calnexin in cargo folding and transport to the ER exit sites

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Leucine Zipper-bearing Kinase promotes axon growth in mammalian central nervous system neurons.

Leucine Zipper-bearing Kinase (LZK/MAP3K13) is a member of the mixed lineage kinase family with high sequence identity to Dual Leucine Zipper Kinase (DLK/MAP3K12). While DLK is established as a key regulator of axonal responses to injury, the role of LZK in mammalian neurons is poorly understood. By gain- and loss-of-function analyses in neuronal cultures, we identify LZK as a novel positive regulator of axon growth. LZK signals specifically through MKK4 and JNKs among MAP2Ks and MAPKs respectively in neuronal cells, with JNK activity positively regulating LZK protein levels. Neuronal maturation or activity deprivation activates the LZK-MKK4-JNK pathway. LZK and DLK share commonalities in signaling, regulation, and effects on axon extension. Furthermore, LZK-dependent regulation of DLK protein expression and the lack of additive effects on axon growth upon co-manipulation suggest complex functional interaction and cross-regulation between these two kinases. Together, our data support the possibility for two structurally related MAP3Ks to work in concert to mediate axonal responses to external insult or injury in mammalian CNS neurons

The unfolded protein response sensor IRE1α is required at 2 distinct steps in B cell lymphopoiesis

B lymphocyte differentiation is coordinated with the induction of high-level Ig secretion and expansion of the secretory pathway. Upon accumulation of unfolded proteins in the lumen of the ER, cells activate an intracellular signaling pathway termed the unfolded protein response (UPR). Two major proximal sensors of the UPR are inositol-requiring enzyme 1α (IRE1α), an ER transmembrane protein kinase/endoribonuclease, and ER-resident eukaryotic translation initiation factor 2α (eIF2α) kinase (PERK). To elucidate whether the UPR plays an important role in lymphopoiesis, we carried out reconstitution of recombinase-activating gene 2–deficient (rag2(–/–)) mice with hematopoietic cells defective in either IRE1α- or PERK-mediated signaling. IRE1α-deficient (ire1α(–/–)) HSCs can proliferate and give rise to pro–B cells that home to bone marrow. However, IRE1α, but not its catalytic activities, is required for Ig gene rearrangement and production of B cell receptors (BCRs). Analysis of rag2(–/–) mice transplanted with IRE1α trans-dominant-negative bone marrow cells demonstrated an additional requirement for IRE1α in B lymphopoiesis: both the IRE1α kinase and RNase catalytic activities are required to splice the mRNA encoding X-box–binding protein 1 (XBP1) for terminal differentiation of mature B cells into antibody-secreting plasma cells. Furthermore, UPR-mediated translational control through eIF2α phosphorylation is not required for B lymphocyte maturation and/or plasma cell differentiation. These results suggest specific requirements of the IRE1α-mediated UPR subpathway in the early and late stages of B lymphopoiesis

ARF6 mediates nephrin tyrosine phosphorylation-induced podocyte cellular dynamics

<div><p>ADP-ribosylation factor 6 (ARF6) is a small GTPase necessary for regulating cellular structure, motility, and vesicle trafficking. In several cellular systems, ARF6 was shown to regulate actin dynamics in coordination with Rac1, a Rho small GTPase. We examined the function of ARF6 in the kidney podocyte because Rac1 was implicated in kidney diseases involving this cell. We found that ARF6 expression was enriched in human podocytes and that it modulated podocyte cytoskeletal dynamics through a functional interaction with nephrin, an intercellular junction protein necessary for podocyte injury-induced signaling requiring activation by tyrosine phosphorylation of its cytoplasmic domain. ARF6 was necessary for nephrin activation-induced ruffling and focal adhesion turnover, possibly by altering Rac1 activity. In podocyte-specific <i>Arf6</i> (ARF6_PodKO) knockout mice, ARF6 deficiency did not result in a spontaneous kidney developmental phenotype or proteinuria after aging. However, ARF6_PodKO mice exhibited distinct phenotypes in two <i>in vivo</i> glomerular injury models. In the protamine sulfate perfusion model, which induced acute podocyte effacement, ARF6_PodKO mice were protected from podocyte effacement. In the nephrotoxic serum nephritis model, which induced immune-complex mediated injury, ARF6_PodKO mice exhibited aggravated proteinuria. Together, these observations suggest that while ARF6 is necessary for nephrin tyrosine phosphorylation-induced cytoskeletal dynamics in cultured podocytes, ARF6 has pleotropic podocyte roles <i>in vivo</i>, where glomerular injury-specific mechanisms might activate distinct signaling pathways that dictate whether ARF6 activity is beneficial or deleterious for maintaining the integrity of the glomerular filtration barrier.</p></div

Nephrin tyrosine phosphorylation increased ARF6 activity and nephrin-ARF6 association.

<p>A. Immunoblot (IB) of total nephrin and tyrosine-phosphorylated nephrin (p-Nephrin) using the CD16/7-nephrin activation model in CD16/7-nephrin expressing human WT podocytes. B. Densitometry analysis of p-nephrin to total nephrin normalized to 0 minutes. Data are shown as mean ± SEM. C. Following nephrin induction in WT podocytes ARF6 activity was assessed by an ARF6 activation assay. GTPγs- and GDP-treated samples were used as positive and negative controls, respectively. D. Densitometry analysis of ARF6-GTP to total ARF6 normalized to 0 minutes. E. CD16/7-nephrin-ARF6 association was assessed by co-immunoprecipitation (IP) where nephrin was immunoprecipitated from podocyte lysates, and nephrin and ARF6 were detected by IB. IP using rabbit IgG was employed as control. F. Densitometry analysis of ARF6 normalized to nephrin. Assays were repeated in triplicate.</p

ARF6KD attenuated nephrin tyrosine phosphorylation-induced Rac1 activity.

<p>A. Following nephrin activation in CD16/7-nephrin expressing human WT or ARF6KD podocytes, Rac1 activity was determined as described in Methods. GTPγs and GDP treated samples were used as positive and negative control, respectively. B. Densitometry analysis of Rac1-GTP to total Rac1 normalized to time 0 minutes.</p

ARF6 is necessary for nephrin ligation-induced ruffling activity.

<p>A. IB of ARF6 protein expression in human WT podocytes, WT podocytes transduced with shRNA control, or <i>ARF6</i> shRNA. B. Densitometry analysis of ARF6 protein abundance normalized to β-actin. C. Percent of podocytes with positive anti-lamellipodin staining following activation of CD16/7-nephrin expressing WT and ARF6 depleted cells. Expression of mutant ARF6 (mtARF6, in which four same-sense mutations were placed within the wild type human ARF6 shRNA target region) was used to rescue ruffling activity in cultured activated CD16/7-nephrin expressing stable ARF6 KD podocytes. D. Percent of podocytes with positive anti-lamellipodin staining 20 minutes following activation of CD16/7 expressing WT, constitutively-active ARF6, ARF6(Q67L), or dominant-negative, ARF6(T27N) podocytes. Approximately 100 cells were evaluated per condition.</p

ARF6KD inhibits nephrin activation-induced dynamic focal adhesion turnover.

<p>A. CD16/7-nephrin (red) expressing WT and stable ARF6-depleted (ARF6KD) human podocytes were used to assess focal adhesion (FA) turnover following CD16/7-nephrin induction. Indirect immunofluorescence microscopy of WT and ARF6KD cells showing CD16/7-nephrin (red) and FA, phosphorylated-paxillin (p-paxillin, green), at indicated time-points following nephrin activation. Magnification 63x. Scale bar 20 μm. B. Quantification of the number of mature FA (≥5 μm Feret’s Diameter) per 100 μm² cell surface area identified by p-paxillin staining at indicated times following nephrin activation. C. Quantification of the number of mature FA (≥5 μm Feret’s Diameter) per 100 μm² cell surface area identified by vinculin staining at indicated times following nephrin activation.</p

Podocyte-specific ARF6 null mice have aggravated proteinuria following nephrotoxic serum nephritis glomerular injury.

<p>A. Urine albumin to creatinine ratio (UACR) was determined for sheep serum or nephrotoxic serum (NTS) treated WT and podocyte-specific ARF6 null mice (mean ± SEM). Results are representative of 10–15 mice per group. B. Day 3 glomerular ultrastructure was assessed by scanning electron microscope. Magnification x3000. Scale bar 5 μm. C. Indirect immunofluorescence of mouse kidney tissue sections stained with anti-WT1 (green) and anti-nephrin (red). Magnification x63. Scale bar 20 μm. D. Podocyte number per glomerular volume (μm³) in all treatment groups.</p