Importance of the Rab3a-GTP Binding Domain for the Intracellular Stability and Function of Rabphilin3a in Secretion

We had previously demonstrated that Rab3a-GTP inhibits and the Rab3a-binding protein Rabphilin3a enhances secretion in bovine chromaffin cells. In this study, we investigated the role of Rab3a-GTP binding in the intracellular expression and the function of Rabphilin3a in regulated exocytosis in bovine chromaffin cells. Using transient transfections, we found that a minimal domain, Rp(51 190), that inhibits secretion coincides with a minimal domain that effectively binds Rab3a-GTP and allows intracellular stability of the construct. This domain includes a cysteine-rich, Zn 2+ -binding domain whose integrity is also required for Rab3a-GTP binding and the ability to inhibit secretion. A Rabphilin3a mutant, containing both C2 domains but defective in Rab3a-GTP, and wild-type Rabphilin3a both localized to chromaffin granules and stimulated secretion similarly despite lessened intracellular expression of the mutant protein. The data are consistent with a sequence of events in which a Rab3a-GTP Rabphilin3a complex forms on the secretory granule as a precursor in a pathway that enhances secretion. The complex dissociates (perhaps because of GTP hydrolysis) to permit the enhancement of secretion by Rabphilin3a.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66356/1/j.1471-4159.1997.69010164.x.pd

Enhancement of BACE1 Activity by p25/Cdk5-Mediated Phosphorylation in Alzheimer's Disease.

The activity of beta-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is elevated during aging and in sporadic Alzheimer's disease (AD), but the underlying mechanisms of this change are not well understood. p25/Cyclin-dependent kinase 5 (Cdk5) has been implicated in the pathogenesis of several neurodegenerative diseases, including AD. Here, we describe a potential mechanism by which BACE activity is increased in AD brains. First, we show that BACE1 is phosphorylated by the p25/Cdk5 complex at Thr252 and that this phosphorylation increases BACE1 activity. Then, we demonstrate that the level of phospho-BACE1 is increased in the brains of AD patients and in mammalian cells and transgenic mice that overexpress p25. Furthermore, the fraction of p25 prepared from iodixanol gradient centrifugation was unexpectedly protected by protease digestion, suggesting that p25/Cdk5-mediated BACE1 phosphorylation may occur in the lumen. These results reveal a link between p25 and BACE1 in AD brains and suggest that upregulated Cdk5 activation by p25 accelerates AD pathogenesis by enhancing BACE1 activity via phosphorylation

p25/Cdk5-mediated phosphorylation of BACE1 increases its activity.

<p>A. Effect of <i>in vitro</i> p25/Cdk5-mediated phosphorylation of BACE1 on its enzymatic activity. B. Effect of a phosphorylation-defective BACE1 mutant (BACE1 T252A) on BACE activity in SK-N-BE(2)C cells. C. Effect of BACE1 T252A on the production of Aβ in HEK293 cells stably expressing APPsw. D. Effect of phosphorylation of Thr252 in BACE1 on basal hGH secretion in PC12 cells. Basal secretion from BACE1 or BACE1 T252A-transfected cells is plotted as a percentage of the secretion from PC12 cells transfected with the control plasmid. Representative immunoblot of extracts from transfected cells with an anti-His antibody is shown below the corresponding graph.</p

Phosphorylation of BACE1 at Thr252 by p25/Cdk5.

<p>A. Coomassie staining (left) and autoradiograph (middle) of SDS polyacrylamide gels containing the products of <i>in vitro</i> kinase assays that employed a BACE1 substrate and the p25/Cdk5 complex. Phospho-BACE1 and phospho-p25 are indicated by an arrow and an arrowhead, respectively. Histone H1, a known substrate of p25/Cdk5, was used as a positive control (right). B. Alignment of human, rat, and mouse BACE1 amino acid sequences. The consensus Cdk5 phosphorylation site is marked in bold. The phosphopeptide used to generate a phosphospecific BACE1 antibody is shown. C. Products of <i>in vitro</i> kinase assays that employed a BACE1 substrate and the p25/Cdk5 complex. Reactions were incubated for the indicated times (left panel) and treated with or without roscovitine, a Cdk5 inhibitor (right panel). Products were then analyzed by western blotting with phosphospecific BACE1 (P-BACE1) antibodies and anti-BACE1 antibodies. D. Western blots for detection of P-BACE1 and BACE1 from HEK293T cells transfected with plasmids encoding the indicated proteins. E. Rat brain lysates were analyzed by western blotting with anti-P-BACE1 antibodies. F. <b>Peptide competition assays demonstrated the specificity of anti-P-BACE1 antibodies.</b> Lysates from HEK293T cells transiently transfected with plasmids encoding p25 and Cdk5 (left panel) or mouse hippocampal lysates (right panel) were analyzed by western blotting with anti-P-BACE1 antibodies pre-incubated in the absence (None) or presence of BACE1-phosphopeptide (SLWYT(PO₄)PIRR), BACE1-nonphosphopeptide (SLWYTPIRR), or p25-phosphopeptide (SAGT(PO₄)PKRVI). G. Co-immunoprecipitation assays showed the interaction between BACE1 and Cdk5. HEK293T cell lysates that were transfected with plasmids encoding p25 and Cdk5 (left panel) or p25 TG mouse hippocampal lysates (right panel) were immunoprecipitated with control IgG or anti-Cdk5 antibodies and then subjected to immunoblot analysis with the indicated antibodies.</p

Phospho-BACE1 is markedly increased in the hippocampus of p25 transgenic (TG) mice.

<p>A. Immunohistochemistry of brain sections from p25 TG mice (20 weeks old, lower panel) and age-matched wild-type (WT) mice (upper panel) using anti-P-BACE1 antibodies. B. Representative western blots of indicated brain regions in two p25 TG mice and control littermates. Arrowheads indicate phospho-BACE1 and the corresponding BACE1 dimer. C. Densitometric analysis of western blots (normalized by β-actin signal) and BACE activity in p25 TG mice plotted as a percent of the WT. D. Aβ amounts in the hippocampus of p25 TG mice plotted as a percent of the WT. Data in C and D are means ± SEMs of 4–7 independent experiments. *<i>p</i> < 0.05 versus WT mice by 2-tailed Student’s t-test.</p

Phospho-BACE1 is cofractionated with p25 and endosome marker in an iodixanol gradient, and fraction 5 of p25 is a luminal protein.

<p>A. Nineteen-month-old p25 TG mouse hippocampus homogenates were separated through an iodixanol step gradient. Western blot analysis showed the distribution of the indicated proteins and organelle markers: Rab5 (early endosome), cathepsin D (lysosome), GM130 (Golgi apparatus), and Bip/Grp78 (ER). B. Two selected fractions, 5 and 10, were digested with PK in the presence or absence of the detergent Triton X-100 (TX-100) followed by immunoblotting analysis with indicated antibodies.</p

Phospho-BACE1 protein is elevated in the superior frontal cortex of human AD brains.

<p>A. Representative western blots of superior frontal cortex tissue (Brodmann area 9) from human AD patients (AD) and normal controls (Con). Arrowheads indicate phospho-BACE1 and the corresponding BACE1 dimer. B. Densitometric analysis of phospho-BACE1 and BACE1 signals in the western blot shown in A, normalized by β-actin signals. Amounts of phospho-BACE1 and BACE1 are plotted as a percent of the control. Control and AD groups consisted of 5 normal (mean age 65 ± 3 years; postmortem interval [PMI], 19 ± 1 h) and 10 AD patients (mean age 69 ± 2 years; PMI, 18 ± 2 h), respectively. *<i>p</i> < 0.05, **<i>p</i> < 0.01 versus the control by Mann-Whitney U test.</p

Association between Skin Cancer and Systemic and Ocular Comorbidities in South Korea

Background: In this study, we investigated the associations between various systemic and ocular comorbidities and skin cancer, in a nationwide cohort of South Koreans. Method: We reviewed the data of 1,103,302 individuals in the South Korean National Health Insurance Service-National Sample Cohort database from 2002 to 2015. Of these, 1202 individuals diagnosed with skin cancer from 2004 were included in the study group. The control group was matched in a 1:5 ratio based on propensity scores. Results: The prevalence rates of melanoma and non-melanoma skin cancer increased from 2004 to 2015. Multivariate logistic regression analysis revealed that, among the various systemic conditions, hypertension was significantly associated with skin cancer, while among ocular comorbidities, macular degeneration showed a significant association with skin cancer. Conclusions: This is the first study to demonstrate associations between skin cancer and various systemic and ocular comorbidities. The results suggest that hypertension and macular degeneration may increase the risk of skin cancer development, or vice versa. Further studies are needed to evaluate the causal relationships between these conditions