Ken-Ichi Kobayashi

Caveolin-1 activates Rab5 and enhances endocytosis through direct interaction.

Caveolin-1, a constitutive protein of the caveolae, is implicated in processes of vesicular transport during caveolae-mediated endocytosis. However, the molecular mechanisms of caveolae-mediated endocytosis are not yet clearly defined. Here, we show the physiological role of the Rab5-caveolin-1 interaction during caveolae-mediated endocytosis. Rab5 was found in caveolae-enriched fractions and Rab5 directly bound to caveolin-1. Furthermore, binding sites of Rab5 to caveolin-1 were identified in the scaffold (SD), transmembrane (TM), and C-terminus (CC) domains, and the Rab5 binding domain of caveolin-1 was required for CTXB uptake. Subsequently, we performed a GST-R5BD pull-down assay to determine whether the Rab5 binding domain of caveolin-1 is involved in Rab5 activity or not. The results showed that overexpression of the Rab5 binding domain of caveolin-1 increase the amount of Rab5-GTP in Cos-1 cells. These findings imply that caveolin-1 controls the Rab5 activity during the caveolae-mediated endocytosis.

Iron deficiency down-regulates the Akt/TSC1-TSC2/mammalian Target of Rapamycin signaling pathway in rats and in COS-1 cells

Iron deficiency (ID) is one of the most commonly known forms of nutritional deficiencies. Low body iron is thought to induce neurologic defects but may also play a protective role against cancer development by cell growth arrest. Thus, ID may affect cellular pathways controlling cell growth and proliferation, the mechanism of which is still not fully understood. The serine/threonine protein kinase Akt and its downstream target, the mammalian Target of Rapamycin (mTOR), is known to play a crucial role in the regulation of cell growth and survival. Therefore, we hypothesized that Akt/mTOR pathway could be influenced by ID. Three-week-old male Wistar-strain rats were divided into 3 groups and the 2 groups had free access to a control diet (C group) or an iron-deficient diet (D group). The third group (PF group) were pair-fed the control diet to the mean intake of the D group. After 4 weeks, rats were killed and their brains were sampled. In separate experiments, COS-1 cells were cultured with or without the iron chelator deferoxamine. Western blots of brain samples and COS-1 lysates were used to analyze the expression and phosphorylation state of Akt, TSC2, mTOR, and S6 kinase proteins implicated in the Akt/mTOR pathway. Using 2 different ID models, we show for the first time that iron deficiency depresses Akt activity in rats and in COS-1 cells, leading to a decrease in mTOR activity.

Interaction of activated rab5 with actin-bundling proteins, l- and t-plastin and its relevance to endocytic functions in mammalian cells

Rab5 is a GTP-binding protein that is crucial for endocytic machinery functions. We previously identified L-plastin as a binding protein for Rab5, using an affinity column with constitutively active Rab5. L- and T-plastin are isoforms of a plastin protein family belonging to actin-bundling proteins that are implicated in the regulation of cell morphology, lamellipodium protrusion, bacterial invasion and tumor progression. However, the physiological relevance of Rab5 binding to plastin has remained unclear. Here, we show that L- and T-plastin interacted only with activated Rab5 and that they co-localized with Rab5 on the plasma membrane and endosome. Rab5 activity was also higher in both L- and T-plastin over-expressing Cos-1 cells. Furthermore, expression of L- and T-plastin increased the rate of fluid-phase endocytosis. These findings imply that the Rab5 is either activated or the activity is sustained by interaction with plastin, and that this interaction influences endocytic activity.

Application of SYPRO Ruby- and Flamingo-stained polyacrylamide gels to Western blot analysis.

Western blot analysis has been a useful method for analysis of expression levels of specific proteins and is conducted after sodium dodecyl sulfate (SDS) or native polyacrylamide gel electrophoresis without staining the gel. However, when it is necessary to analyze the gel, duplicate polyacrylamide gels usually must be prepared, one of which is stained, leading to the consumption of precious sample. Thus, we developed a convenient and efficient Western blotting method using a stained gel. This simple modification should be beneficial for analyzing samples that are limited in quantity and/or samples for which the stained gel serves as the loading control.

Activation of Nrf2/Keap1 signaling and autophagy induction against oxidative stress in heart in iron deficiency

We investigated the effects of dietary iron deficiency on the redox system in the heart. Dietary iron deficiency increased heart weight and accumulation of carbonylated proteins. However, expression levels of heme oxygenase-1 and LC3-II, an antioxidant enzyme and an autophagic marker, respectively, in iron-deficient mice were upregulated compared to the control group, resulting in a surrogate phenomenon against oxidative stress.

Rab5 affinity chromatography without nonhydrolyzable GTP analogues.

Rab5 is an important small GTPase involved in endocytosis and membrane trafficking. Rab5-binding proteins can be identified using Rab5 affinity chromatography with nonhydrolyzable GTP analogues such as GTPγS or GppNHp. However, this method requires significant quantities of the GTP analogue and is thus time-consuming and expensive. In the present report we show a faster and more cost-effective method that does not use a GTP analogue but uses constitutively the active Rab5 mutant (Rab5Q79L) as a ligand. To validate this method, the binding of EEA-1 was confirmed and several novel Rab5-binding proteins were also identified by 2-dimensional electrophoresis and liquid chromatographymass spectrometry/mass spectrometry (LC-MS/MS).

Hamartin-Hsp70 Interaction Is Necessary for Akt-Dependent Tuberin Phosphorylation during Heat Shock

Hamartin and tuberin interact directly to regulate cell growth negatively. In this study, far-western blotting revealed that hamartin binds directly Heat shock protein 70 (Hsp70), even in the absence of tuberin. While the hamartin-tuberin complex acts as a sensor for a variety of types of stress, it is unclear how the complex is regulated under stress conditions. We found that the hamartin-Hsp70 interaction is stabilized during heat shock. On the other hand, tuberin underwent degradation through phosphorylation in an Akt-dependent manner. Furthermore, we found that when Hsp70 expression was inhibited by N-formyl-3,4-methylenedioxy-benzylidene-γ-butyrolactam (KNK437), Akt phosphorylation on site Ser308 diminished and tuberin was not phosphorylated at Thr1462 during heat shock. We conclude that both hamartin and Hsp70 increase in response to heat shock, whereas tuberin is phosphorylated and thereafter degraded via the PI3K/Akt pathway. Through this pathway, hamartin-Hsp70 plays a crucial role as a scaffolding protein that transfers the Akt signal to tuberin.

High dietary fat-induced obesity in Wistar rats and type 2 diabetes in nonobese Goto-Kakizaki rats differentially affect retinol binding protein 4 expression and vitamin A metabolism

Obesity is a major risk factor for type 2 diabetes, which is caused mainly by insulin resistance. Retinol binding protein 4 (RBP4) is the only specific transport protein for retinol in the serum. RBP4 level is increased in the diabetic state and high-fat condition, indicating that retinol metabolism may be affected under these conditions. However, the precise effect of diabetes and high fat-induced obesity on retinol metabolism is unknown. In this study, we examined differences in retinol metabolite levels in rat models of diet-induced obesity and type 2 diabetes (Goto-Kakizaki [GK] rat). Four-week-old male Wistar and GK rats were given either a control diet (AIN-93G) or a high-fat diet (HFD, 40% fat kJ). After 15 weeks of feeding, the RBP4 levels increased by 2-fold in the serum of GK rats but not HFD-fed rats. The hepatic retinol concentration of HFD-fed rats was approximately 50% that of the controls (P < .01). In contrast, the renal retinol concentrations of GK rats increased by 70% (P < .01). However, expression of RARβ in the kidney, which was induced in a retinoic acid-dependent manner, was downregulated by 90% (P < .01) in GK rats. In conclusion, diabetes and obesity affected retinol metabolism differently, and the effects were different in different peripheral tissues. The impact of HFD may be limited to the storage of hepatic vitamin A as retinyl palmitate. In particular, our data indicate that renal retinoic acid production might represent an important target for the treatment of type 2 diabetes mellitus.

N-Formyl-3,4-methylenedioxy-benzylidene-γ-butyrolaetam, KNK437 induces caspase-3 activation through inhibition of mTORC1 activity in Cos-1 cells

The mammalian target of rapamycin complex 1 (mTORC1: mTOR-raptor interaction) and heat shock protein 70 (Hsp70) regulate various cellular processes and are crucial for the progression of many cancers and metabolic diseases. In the recent study, we reported that interaction of Hsp70 with tuberous sclerosis complex 1 (TSC1) regulated apoptosis. This study was designed to elucidate the underlying mechanism in Cos-1 cells. Here, we show that N-formyl-3,4-methylenedioxy-benzylidene-gamma-butyrolaetam (KNK437), which inhibits the expression level of Hsp70, abrogated phosphorylation of mTOR and S6K in response to insulin, and inhibited mTORC1 activity via disruption of an interaction between mTOR and raptor. In addition, KNK437 did not alter TSC1/2 complex formation. Furthermore, KNK437 inhibited the mTOR-raptor interaction on the outer membrane of the mitochondria and triggered caspase-3 activation. A reduction in the level of Hsp70 could result in the inhibition of the mTORC1 signaling pathway, thereby inducing apoptosis.

Phosphorylated hamartin–Hsp70 complex regulates apoptosis via mitochondrial localization

The products of the tuberous sclerosis complex (TSC) genes, hamartin and tuberin, form a heterodimer. Recently we reported that hamartin directly interacted with Hsp70. However, the physiological implications of this interaction have not yet been clearly defined. Here we show that hamartin localized to the outer membrane of the mitochondria in an Hsp70-dependent manner. Moreover, phosphorylation of the T417 residue of hamartin was required for its localization to the mitochondria as well as its interaction with Hsp70. A non-phosphorylatable hamartin mutant at residue T417 was unable to localize to the mitochondria and suppress apoptosis, whereas non-phosphorylatable hamartin mutants T357A and T390A localized to the mitochondria and suppressed apoptosis. Importantly, non-phosphorylatable mutants (T357A, T390A and T417A) promoted apoptosis after treatment with Hsp 70-inhibitor KNK437. We conclude that hamartin inhibited apoptosis by localizing to the mitochondria and that its phosphorylation and binding to Hsp70 was required for facilitation of this process.