Identification of Nedd4 E3 Ubiquitin Ligase as a Binding Partner and Regulator of MAK-V Protein Kinase

MAK-V/Hunk is a scantily characterized AMPK-like protein kinase. Recent findings identified MAK-V as a pro-survival and anti-apoptotic protein and revealed its role in embryonic development as well as in tumorigenesis and metastasis. However molecular mechanisms of MAK-V action and regulation of its activity remain largely unknown. We identified Nedd4 as an interaction partner for MAK-V protein kinase. However, this HECT-type E3 ubiquitin ligase is not involved in the control of MAK-V degradation by the ubiquitin-proteasome system that regulates MAK-V abundance in cells. However, Nedd4 in an ubiquitin ligase-independent manner rescued developmental defects in Xenopus embryos induced by MAK-V overexpression, suggesting physiological relevance of interaction between MAK-V and Nedd4. This identifies Nedd4 as the first known regulator of MAK-V function

Pathogenetics of alveolar capillary dysplasia with misalignment of pulmonary veins.

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a lethal lung developmental disorder caused by heterozygous point mutations or genomic deletion copy-number variants (CNVs) of FOXF1 or its upstream enhancer involving fetal lung-expressed long noncoding RNA genes LINC01081 and LINC01082. Using custom-designed array comparative genomic hybridization, Sanger sequencing, whole exome sequencing (WES), and bioinformatic analyses, we studied 22 new unrelated families (20 postnatal and two prenatal) with clinically diagnosed ACDMPV. We describe novel deletion CNVs at the FOXF1 locus in 13 unrelated ACDMPV patients. Together with the previously reported cases, all 31 genomic deletions in 16q24.1, pathogenic for ACDMPV, for which parental origin was determined, arose de novo with 30 of them occurring on the maternally inherited chromosome 16, strongly implicating genomic imprinting of the FOXF1 locus in human lungs. Surprisingly, we have also identified four ACDMPV families with the pathogenic variants in the FOXF1 locus that arose on paternal chromosome 16. Interestingly, a combination of the severe cardiac defects, including hypoplastic left heart, and single umbilical artery were observed only in children with deletion CNVs involving FOXF1 and its upstream enhancer. Our data demonstrate that genomic imprinting at 16q24.1 plays an important role in variable ACDMPV manifestation likely through long-range regulation of FOXF1 expression, and may be also responsible for key phenotypic features of maternal uniparental disomy 16. Moreover, in one family, WES revealed a de novo missense variant in ESRP1, potentially implicating FGF signaling in the etiology of ACDMPV

MAK-V is not ubiquitinated by Nedd4.

<p>(<b>A</b>) E2 enzyme specificity of ubiquitin-ligase activity co-purified with MAK-V-FLAG protein. MAK-V-FLAG protein purified from PC12TetOn cells was used in <i>in vitro</i> ubiquitination reactions containing E1 enzyme and indicated E2 ubiquitin-conjugating enzymes. Results of Western blotting with HRP-conjugated streptavidin to detect ubiquitinated proteins/polyubiquitin (marked by asterisk) are shown. The image was overlaid with an image of the same membrane that was consequently probed with anti-FLAG antibodies to detect MAK-V-FLAG protein (marked by arrows). (<b>B</b>) Depletion of Nedd4 does not affect stabilization of MAK-V-FLAG protein in cells in response to treatment with proteasome inhibitors. Parental PC12TetOn cells with inducible MAK-V FLAG expression (<i>no miR</i>) or their clonal derivatives expressing control microRNA (<i>control mi</i>R) or microRNA to target Nedd4 transcript (<i>Nedd4 miR</i>) were treated with doxycycline to express MAK-V-FLAG protein and incubated with 100 µM of ALLN (<i>ALLN</i>) or 10 µM of MG132 (<i>MG132</i>) for 8 hrs or left untreated (-). Results of Western blot analysis of total cell lysates with anti-FLAG antibodies are shown (<i>anti-FLAG</i>). Nedd4 level was monitored with anti-Nedd4 antibodies (<i>anti-Nedd4</i>). MAK-V-FLAG protein marked with arrow, ubiquitinated higher molecular weight MAK-V-FLAG species marked by asterisk. Membrane was also probed with anti-α-tubulin antibodies (<i>anti-α-tubulin</i>) to monitor total protein loading. (<b>C</b>) MAK-V-FLAG protein and cytosols were prepared from parental PC12TetOn cells with inducible MAK-V-FLAG expression (<i>Nedd4</i>+) or its Nedd4-depleted clonal derivative expressing Nedd4 microRNA (<i>Nedd4</i> -) and used in <i>in vitro</i> ubiquitination reaction containing E1 and UbcH5b E2 enzymes. Results of Western blot analysis with anti-FLAG antibodies are shown. Arrow marks products of MAK-V ubiquitination. Reactions in the presence of EDTA (<i>EDTA</i> +) were used as negative controls.</p

Nedd4 regulates MAK-V activity.

<p>Nedd4 in ubiquitin ligase-independent manner rescue axis extension defects in <i>Xenopus</i> development induced by MAK-V overexpression. Two ng each of <i>MAK-V</i>, <i>Nedd4</i>, <i>Nedd4(CS)</i> mRNA was injected alone or together as indicated into two dorso-animal blastomeres at 4–8 cell stage. Embryos were fixed at stage 38.</p

Nedd4 specifically interacts with MAK-V.

<p>(<b>A</b>) Typical profile of proteins purified on anti-FLAG M2 affinity gel from PC12TetOn cells treated with doxycycline (+) to express MAK-V-FLAG protein or left untreated (-). Silver-stained 4–12% gel is shown with positions of protein molecular weight markers (MW) shown in kD on the left. Arrows marks MAK-V-FLAG protein (*) and Nedd4 protein co-purified with MAK-V-FLAG (**). (<b>B</b>) Samples from (<b>A</b>) (<i>IP anti-FLAG</i>) and aliquots of cell lysates prior to purification (<i>Input</i>) were stained with anti-Nedd4 (<i>anti-Nedd4</i>) or anti-FLAG (<i>anti-FLAG</i>) antibodies. (<b>C</b>) Lysate of PC12TetOn cells treated with doxycycline to express MAK-V-FLAG protein was incubated with Protein G Sepharose (<i>no Ab</i>) or Protein G Sepharose with immobilized anti-Nedd4 antibodies (<i>Nedd4 Ab</i>). Bound proteins were analyzed by Western blotting with anti-FLAG (<i>anti-FLAG</i>) or anti-Nedd4 (<i>anti-Nedd4</i>) antibodies. (<b>D</b>) Proteins were pulled down from lysate of PC12TetOn cells treated with doxycycline to express MAK-V-FLAG protein with GST (<i>GST</i>) or GST-Nedd4 (<i>GST-Nedd4</i>) proteins immobilized on glutathione Sepharose. MAK-V-FLAG protein was detected with anti-FLAG antibodies (<i>anti-FLAG</i>). To monitor GST protein loading of glutathione Sepharose, eluted proteins were stained with anti-GST antibodies (<i>anti-GST</i>). Arrowhead marks full-length GST-Nedd4 chimeric protein.</p

Nedd4 does not affect MAK-V protein level.

<p>(<b>A</b>) HEK293 cells were transfected with MAK-V-FLAG expression plasmid (<i>MAK-V-FLAG +</i>) together with plasmid for expression of <i>myc</i>-tagged wild-type Nedd4 (<i>wt</i>) or its catalytically inactive Nedd4(CS) mutant (<i>CS</i>), or mock-untransfected (-). Cells were treated by MG132 prior to lysis (<i>MG132+</i>) or left untreated (<i>MG132 -</i>). Results of Western blot analysis of total cell lysates with anti-FLAG antibodies are shown (<i>anti-FLAG</i>). Nedd4 levels were monitored with anti-Nedd4 antibodies (<i>anti-Nedd4</i>), and exogenously expressed myc-tagged Nedd4 proteins were detected with anti-<i>myc</i> antibodies (<i>anti-myc</i>). To monitor total protein loading, membrane was re-probed with anti-α-tubulin antibodies. (<b>B</b>) HEK293 cells were co-transfected with MAK-V-FLAG expression plasmid and plasmid for expression of <i>myc</i>-tagged wild-type Nedd4 (<i>Nedd4 wt</i>) or its catalytically inactive Nedd4(CS) mutant (<i>Nedd4(CS</i>)). Cells were treated with cycloheximide for indicated time prior to lysis. Representative results of Western blot analysis of total cell lysates with anti-FLAG antibodies to detect MAK-V-FLAG protein are shown (<i>anti-FLAG</i>). To monitor total protein loading, membrane was re-probed with anti-α-tubulin antibodies. (<b>C</b>) Quantification of MAK-V-FLAG protein level in HEK293 cells when co-expressed with wild-type Nedd4 (<i>Nedd4 wt</i>) or with its Nedd4(CS) (<i>Nedd4 CS</i>) mutant after cycloheximide treatment. Relative MAK-V-FLAG protein levels for each time point of cycloheximide treatment indicated in hours (<i>hrs</i>) were determined by quantification of Western blot images obtained as described in (<b>B</b>). Data are presented as means ± standard deviation of three independent experiments performed as described in (<b>B</b>).</p

MAK-V is subjected to ubiquitin-dependent proteasomal degradation.

<p>(<b>A</b>) Doxycycline-treated (<i>DOX +</i>) or untreated (<i>DOX</i> -) PC12TetOn MAK-V-FLAG cells were incubated with 100 µM of ALLN (<i>ALLN</i> +) or vehicle (<i>ALLN</i> -) for 8 hrs. Results of Western blot analysis of total cell lysates with anti-FLAG antibodies are shown. MAK-V-FLAG protein marked with arrow, ubiquitinated higher molecular weight MAK-V-FLAG species marked by asterisk. To monitor total protein loading, membrane was re- probed with anti-α-tubulin antibodies. (<b>B</b>) HEK293 cells were transiently transfected with plasmid for MAK-V-FLAG protein expression and treated with 10 µM MG132 (<i>MG+</i>) for indicated time prior to lysis or left untreated (<i>MG-</i>). Lysates were blotted with anti-FLAG antibodies to detect MAK-V-FLAG protein (<i>anti-FLAG</i>). To monitor total protein loading, membrane was re-probed with anti-α-tubulin antibodies. (<b>C</b>) HEK293 cells were transfected with plasmid for HA-tagged ubiquitin expression alone (<i>MAK-V-FLAG -</i>) or together with plasmid for MAK-V-FLAG protein expression (<i>MAK-V-FLAG +</i>). Cells were treated with MG132 prior to lysis. MAK-V-FLAG protein was precipitated from lysates with anti-FLAG M2 affinity gel as described for PC12TetOn cells. Anti-FLAG antibodies were used to detect MAK-V-FLAG protein (<i>anti-FLAG</i>) in immunoprecipitates (<i>IP: anti-FLAG</i>), and ubiquitin was detected with anti-HA antibodies (<i>anti-HA</i>). To monitor HA-tagged ubiquitin and MAK-V-FLAG protein expression, aliquots of lysates were blotted as described above. To monitor total protein loading, membrane was re-probed with anti-α-tubulin antibodies. (<b>D</b>) HEK293 cells were co-transfected with MAK-V-FLAG expression plasmid and plasmid for expression of FLAG-tagged wild-type ubiquitin (<i>wt</i>), its K48R (<i>K48R</i>) or K63R (<i>K63R</i>) mutant. Results of Western blot analysis of total cell lysates with anti-MAK-V antibodies are shown. To monitor total protein loading, membrane was re-probed with anti-α-tubulin antibodies. To control variations in basal level of MAK-V-FLAG protein expression, samples were also probed with antibodies against neomycin phosphotransferase II (<i>anti-NPT</i>) which is encoded by MAK-V-FLAG expression vector.</p

Interaction between endogenous Nedd4 and MAK-V proteins.

<p>Proteins were immunoprecipitated from lysates of CSML-0 cells with anti-Nedd4 (<b>A</b>; <i>IP: Nedd4 Ab</i>) or anti-MAK-V antibodies (<b>B</b>; <i>IP: MAK-V Ab</i>). Control immunoprecipitations with omitted antibodies were run in parallel (<i>IP: control</i>). Immunoprecipitates and aliquots of lysates used for immunoprecipitation (<i>Input</i>) were probed with anti-MAK-V (<i>anti-MAK-V</i>) and anti-Nedd4 (<i>anti-Nedd4</i>) antibodies to detect MAK-V and Nedd4 proteins, respectively.</p