Phosphorylation of p62 at S409 is required for autophagic degradation of polyubiquitinated proteins and the recruitment of autophagy proteins.

<p><b>A.-C</b>. p62 p-S409 regulates autophagic degradation of ubiquitinated proteins. p62 KO MEFs stably expressing indicated constructs were treated with MG132 for 16 hr(pre), then media were switched to serum starvation(S.S.), in combination with CQ for 24 hr(post). <b>A</b>. p62 S409A overexpressing cells are resistant to autophagic degradation of poly-Ub proteins. Total cellular lysates were subjected to immunoblot assay with indicated antibodies. <b>B</b>. The level of poly-Ub proteins from Fig. 7A was normalized to actin level, and further to each own control. Student’s <i>t</i>-test was used and data are represented as mean ± SEM(n = 4). * <i>p</i> < 0.05; ns, not significant <b>C</b>. p62 S409A is impaired in the recruitment of autophagy machinery proteins. MEFs were fixed, stained with antibodies against p62(red) and WIPI2, LC3, Rab7, or LAMP2(green), and then visualized under fluorescent microscope. Line profile was used to illustrate co-localization. Green lines indicate WIPI2, LC3, Rab7, or LAMP2 and red lines indicate p62. Scale bar = 10 μm, 5 μm for enlarged images.</p

ULK1-mediated phosphorylation of p62 at S409 enhances p62 and Ub binding affinity.

<p><b>A</b>. P-S409 enhances binding between p62 and poly-Ub proteins. Cellular lysates of p62 KO MEFs stably expressing empty vector, FLAG-p62 WT, S409A, or S409E were incubated with p62 KO MEFs lysates treated with MG132 and subsequently subjected to IP using anti-FLAG antibody. Immunoblot assay with indicated antibodies was followed. <b>B</b>. Quantification of the results from Fig. 5A were obtained by normalizing levels of IPed Ub to FLAG blots; then S409A or S409E were normalized to WT. One sample <i>t</i>-test was used and data are represented as mean ± SEM(n = 3). * <i>p</i> < 0.05; ns, not significant <b>C</b>. p62 UBA S409E has an enhanced binding affinity to mono-Ub. Binding affinities of p62 UBA WT(left) or S409E(right) to mono-Ub were measured by Isothermal Titration Calorimetry(ITC). Representative ITC profiles are shown.</p

Phosphorylation of p62 at S409 enhances the autophagic degradation of polyQ-Htt mutant proteins.

<p>HeLa/65Q–mCFP cells were transfected with mCherry-p62 WT, S409A or S409E and treated with Rapamycin to induce autophagy. <b>A</b>. Cells were fixed and visualized under fluorescent confocal microscope. Scale bar = 20 μm. <b>B</b>. Quantifications of the results in Fig. 8A were performed by counting cell numbers containing 65Q-mCFP aggregates. Then the number of cell carrying 65Q-mCFP aggregates was normalized to the number of control cells transfected with p62 WT(left panel). Efficiency of polyQ clearance(right panel) was obtained from the ratio of control sample vs. rapamycin-treated sample. <b>C</b>. Cells were separated into detergent soluble and insoluble fractions and probed with indicated antibodies. <b>D</b>. Quantifications were performed by normalizing the intensity to that of control samples transfected with p62 WT(upper panel). Efficiency of polyQ reduction(lower panel) based on the results in Fig. 8C was obtained as described in Fig. 8B. One sample <i>t</i>-test and student <i>t</i>-test were used and data are represented as mean ± SEM(n = 4). * <i>p</i> < 0.05, ** <i>p</i> < 0.01; ns, not significant.</p

The working model for ULK1-mediated p-S409 and p-S405 of p62 in selective degradation of ubiquitinated proteins and polyglutamine-expanded proteins.

<p>Accumulated polyubiquitinated(poly-Ub) proteins or polyQ-expanded proteins trigger interaction of p62 with ULK1. This interaction induces ULK1-mediated p62 phosphorylation at S409 in UBA domain, which facilitates dimer to monomer transition of UBA domain, and subsequent phosphorylation at S405(mediated by either ULK1, CK2 or TBK-1). As a result, the phosphorylation of p62 at S405 and S409 leads to enhanced binding affinity of p62 to poly-Ub or polyQ-expanded proteins. The presence of ULK1 and p62 p-S405 and p-S409 in poly-Ub or polyQ-expanded protein aggregates causes the recruitment of autophagy machinery that is responsible for the degradation of poly-Ub or polyQ-Htt mutant proteins.</p

ULK1 phosphorylates p62 at S409.

<p><b>A.-C</b>. <i>In vitro</i> p62 phosphorylation assay by ULK1 with purified MBP-p62 WT or mutant proteins. Bacterially expressed MBP-p62 was purified and then MBP tag was cleaved by Factor Xa. The purified p62 was incubated with Myc-ULK1 WT or KI mutant IPed from transfected HEK293T cells at 37°C for 30 min. Phosphorylation of p62 was examined by ³²P-labeling and autoradiography or p-S409 specific antibody. <b>A</b>. p62 is an ULK1 substrate <i>in vitro</i>. Alkaline phosphatase(AP) was used to dephosphorylate p62. ³²P-autoradiograph shows autophosphorylation of ULK1 and p62 phosphorylation. <b>B</b>. ULK1 phosphorylates p62 at Ser409 <i>in vitro</i>. Purified MBP-p62 WT or S409A proteins were used in ULK1 kinase assay in the presence of ³²P-ATP. <b>C</b>. ULK1 phosphorylates Ser409 of p62. Purified MBP-62 WT proteins were incubated with Myc-ULK1 variants isolated from transfected HEK 293T cells. Immunoblotting assay with indicated antibodies, including phospho-p62 antibody against Ser409, was followed. Afterwards, the membrane probed with p-p62 antibody(S409) was incubated with alkaline phosphatase(AP) to dephosphorylate p62. Asterisks indicate nonspecific bands. <b>D</b>. p62 S409 is a ULK1 substrate. HEK 293T cells were transfected wit empty vector, FLAG-p62 WT or FLAG-p62 S409A together with Myc-ULK1 WT or KI. IP with anti-FLAG antibody was performed, followed by Westernblot assay with indicated antibodies.</p

Phosphorylation of p62 at S409 is enhanced upon proteasome inhibition.

<p><b>A</b>. ULK1 mediates p-S409 of p62 upon MG132 treatment. HEK 293T cells transfected with empty vector, Myc-ULK1 WT or KI were treated with MG132 and then cell lysates were analysed with indicated antibodies(Top). The ratio of p-p62 and p62 was obtained by dividing the level of p-p62 by total p62(n = 3). One sample <i>t</i>-test was used and data are represented as mean ± SEM(n = 3). * <i>p</i> < 0.05; ns, not significant(bottom). <b>B</b>. ULK1 is important for MG132-induced p-S409 of p62. WT and ULK1 KO MEFs were treated with MG132 and analyzed with indicated antibodies(Top). The ratio of p-p62 and p62 was obtained by dividing the level of p-p62 by total p62(n = 3). One sample <i>t</i>-test was used and data are represented as mean ± SEM(n = 3). * <i>p</i> < 0.05; ns, not significant(bottom). <b>C</b>. MG132 treatment but not starvation increases p-S409 levels of p62 or ULK1-p62 interaction. p62 KO MEFs stably expressing empty vector or FLAG-p62 were incubated with MG132 or starved for glucose(-glu) or amino acid(-aa). F.M. indicates full medium as a control. Subsequently, IP with anti-FLAG or-ULK1 antibodies were performed and immunoprecipitants were analyzed with indicated antibodies.</p

DataSheet_1_Associations between exercise capacity, p16INK4a expression and inflammation among adult survivors of childhood cancer.pdf

BackgroundOver 50% of childhood cancer survivors are exercise intolerant, with maximal aerobic capacities comparable to individuals decades older, suggesting early physiologic ageing. In addition, 36% of survivors are obese. Optimal exercise capacity provides a foundation to support daily function and healthy body habitus and is associated with benefits to cognition, cardiovascular health, and longevity. Cellular senescence and inflammation are key mechanisms that drive age-related disease, quantifiable as biomarkers in peripheral blood.AimsThis study aimed to evaluate associations between p16INKa, a biomarker of cellular senescence, and inflammation and exercise capacity among adult survivors of childhood cancer.Materials and methodsEligible survivors were recruited from the St. Jude Lifetime (SJLIFE) Cohort Study. Exercise capacity was assessed by maximal oxygen uptake (VO2, ml/kg/min) obtained via cardiopulmonary exercise testing using a modified Bruce protocol. Body fat (%) was determined from dual energy x-ray absorptiometry (DEXA). Peripheral blood samples were used to evaluate log2 p16INK4a mRNA expression, a biomarker of cellular senescence, and inflammation with high sensitivity C-reactive protein (hs-CRP) levels. Multivariable regression evaluated associations between p16INK4a, hs-CRP, body fat, and exercise capacity.ResultsParticipants included 185 five-year childhood cancer survivors (mean age 36.6 [range 20.1 - 55.7] years, 44% male, 77% non-Hispanic white, 53% leukemia/lymphoma). Compared to males, females had lower peak VO2 (mean ± SD, 22.5 ± 8.2 vs. 28.8 ± 7.7 ml/kg/min, pINK4a expression (9.6 ± 1.2 vs. 9.2 ± 1.2 fold, p=0.02), and hs-CRP concentration (5.9 ± 8.4 vs. 3.3 ± 3.9 mg/L, p=0.01). Among females (n=103), hs-CRP concentration (β -0.2, 95% CI -0.34 to -0.05, p=0.01) and p16INK4a expression (β-5.32, 95% CI 10.42 to -0.22, p=0.04) were inversely associated and statistically significant with peak exercise capacity, with a significant interaction between p16INK4a expression and body fat (β 0.15, 95% CI 0.02 to 0.28, p=0.03). Among males (n=82), p16INK4a expression (β -1.01, 95% CI -2.14 to 0.12, p=0.08), and body fat (β -0.54, 95% CI -0.70 to -0.38, pConclusionInflammation and p16INK4a expression, a biomarker of cellular senescence, are associated with lower exercise capacity in childhood cancer survivors, suggesting potential targets or outcome measures for interventions designed to prevent or remediate accelerated physiologic ageing in this population.</p

The autophagy-inducing kinases, ULK1 and ULK2, regulate axon guidance in the developing mouse forebrain via a noncanonical pathway

<p>Mammalian ULK1 (unc-51 like kinase 1) and ULK2, <i>Caenorhabditis elegans</i> UNC-51, and <i>Drosophila melanogaster</i> Atg1 are serine/threonine kinases that regulate flux through the autophagy pathway in response to various types of cellular stress. <i>C. elegans</i> UNC-51 and <i>D. melanogaster</i> Atg1 also promote axonal growth and defasciculation; disruption of these genes results in defective axon guidance in invertebrates. Although disrupting ULK1/2 function impairs normal neurite outgrowth in vitro, the role of ULK1 and ULK2 in the developing brain remains poorly characterized. Here, we show that ULK1 and ULK2 are required for proper projection of axons in the forebrain. Mice lacking <i>Ulk1</i> and <i>Ulk2</i> in their central nervous systems showed defects in axonal pathfinding and defasciculation affecting the corpus callosum, anterior commissure, corticothalamic axons and thalamocortical axons. These defects impaired the midline crossing of callosal axons and caused hypoplasia of the anterior commissure and disorganization of the somatosensory cortex. The axon guidance defects observed in <i>ulk1/2</i> double-knockout mice and central nervous system-specific (<i>Nes-Cre</i>) <i>Ulk1/2</i>-conditional double-knockout mice were not recapitulated in mice lacking other autophagy genes (i.e., <i>Atg7</i> or <i>Rb1cc1</i> [RB1-inducible coiled-coil 1]). The brains of <i>Ulk1/2</i>-deficient mice did not show stem cell defects previously attributed to defective autophagy in <i>ambra1</i> (autophagy/Beclin 1 regulator 1)- and <i>Rb1cc1</i>-deficient mice or accumulation of SQSTM1 (sequestosome 1)⁺ or ubiquitin⁺ deposits. Together, these data demonstrate that ULK1 and ULK2 regulate axon guidance during mammalian brain development via a noncanonical (i.e., autophagy-independent) pathway.</p