Jiro Toshima

Binding of 14-3-3β Regulates the Kinase Activity and Subcellular Localization of Testicular Protein Kinase 1

Testicular protein kinase 1 (TESK1) is a serine/threonine kinase that phosphorylates cofilin and induces actin cytoskeletal reorganization. The kinase activity of TESK1 is stimulated by integrin-mediated signaling pathways, but the mechanism of regulation has remained unknown. By using the yeast two-hybrid system, we identified 14-3-3β to be the binding protein of TESK1. Specific interaction between TESK1 and 14-3-3β became evident in in vitro and in vivo co-precipitation assays. 14-3-3β interacts with TESK1 through the C-terminal region of TESK1 and in a manner dependent on the phosphorylation of Ser-439 within an RXXSXP motif. Binding of 14-3-3β inhibited the kinase activity of TESK1. During cell spreading on fibronectin, the TESK1/14-3-3β interaction significantly decreased, in a time course that inversely correlated with increase in TESK1 kinase activity. Thus, the dissociation of 14-3-3β from a TESK1/14-3-3β complex is likely to be involved in the integrin-mediated TESK1 activation. In HeLa cells, TESK1, together with 14-3-3β, accumulated at the cell periphery when cells were plated on fibronectin, whereas they were diffusely distributed in the cytoplasm in the case of non-stimulated cells. We propose that 14-3-3β plays important roles in regulating the kinase activity of TESK1 and localizing TESK1 to cell adhesion sites following integrin stimulation.

Sprouty-4 negatively regulates cell spreading by inhibiting the kinase activity of testicular protein kinase

TESK1 (testicular protein kinase 1) is a serine/threonine kinase that phosphorylates cofilin and plays a critical role in integrin-mediated actin cytoskeletal reorganization and cell spreading. We previously showed that TESK1 interacts with Sprouty-4 (referred to as Spry4), an inhibitor of growth factor-induced Ras/MAP (mitogen-activated protein) kinase signalling, but the functional role of this interaction has remained unknown. In the present study, we show that Spry4 inhibits the kinase activity of TESK1 by binding to it through the C-terminal cysteine-rich region. Expression of Spry4 in cultured cells suppressed integrin-mediated cell spreading, and TESK1 reversed the inhibitory effect of Spry4 on cell spreading. Furthermore, Spry4 suppressed integrin- and TESK1-mediated cofilin phosphorylation during the spreading of cells on laminin. These findings suggest that Spry4 suppresses cell spreading by inhibiting the kinase activity of TESK1. Although tyrosine phosphorylation is required for the inhibitory activity of Spry4 on a Ras/MAP kinase pathway, mutation of the corresponding tyrosine residue (Tyr-75 in human Spry4) to an alanine had no apparent effect on its inhibitory actions on TESK1 activity and cell spreading, which suggests a novel cellular function of Spry to regulate the actin cytoskeleton, independent of its inhibitory activity on the Ras/MAP kinase signalling.

Requirements for recruitment of a G protein-coupled receptor to clathrin-coated pits in budding yeast

Endocytic internalization of G protein-coupled receptors (GPCRs) plays a critical role in down-regulation of GPCR signaling. The yeast mating pheromone receptor Ste2p has been used as a model to investigate mechanisms of signal transduction, modification, and endocytic internalization of GPCRs. We previously used a fluorescently labeled mating pheromone derivative to reveal unappreciated molecular and spatiotemporal features of GPCR endocytosis in budding yeast. Here, we identify recruitment of Ste2p to preexisting clathrin-coated pits (CCPs) as a key step regulated by receptor phosphorylation and subsequent ubiquitination upon ligand binding. The yeast casein kinase I homologue Yck2p directly phosphorylates six serine residues located in the C-terminal tail of Ste2p, and mutation of these serine residues to alanine significantly decreased recruitment of Ste2p to CCPs. We also found that the clathrin adaptors Ent1p, Ent2p, and Ede1p work cooperatively to recruit ubiquitinated Ste2p to CCPs. In addition, ubiquitination has a role in ligand-independent constitutive recruitment of Ste2p to CCPs, although this process is much slower than ligand-induced recruitment. These results suggest that ubiquitination of Ste2p is indispensable for recruiting Ste2p to CCPs in both ligand-dependent and ligand-independent endocytosis.

Dual specificity protein kinase activity of testis-specific protein kinase 1 and its regulation by autophosphorylation of serine-215 within the activation loop.

TESK1 (testis-specific protein kinase 1) is a protein kinase with a structure composed of an N-terminal protein kinase domain and a C-terminal proline-rich domain. Whereas the 3.6-kilobase TESK1 mRNA is expressed predominantly in the testis, a faint 2.5-kilobase TESK1 mRNA is expressed ubiquitously. The kinase domain of TESK1 contains in the catalytic loop in subdomain VIB an unusual DLTSKN sequence, which is not related to the consensus sequence of either serine/threonine kinases or tyrosine kinases. In this study, we show that TESK1 has kinase activity with dual specificity on both serine/threonine and tyrosine residues. In an in vitrokinase reaction, the kinase domain of TESK1 underwent autophosphorylation on serine and tyrosine residues and catalyzed phosphorylation of histone H3 and myelin basic protein on serine, threonine, and tyrosine residues. Site-directed mutagenesis analyses revealed that Ser-215 within the “activation loop” of the kinase domain is the site of serine autophosphorylation of TESK1. Replacement of Ser-215 by alanine almost completely abolished serine autophosphorylation and histone H3 kinase activities. In contrast, replacement of Ser-215 by glutamic acid abolished serine autophosphorylation activity but retained histone H3 kinase activity. These results suggest that autophosphorylation of Ser-215 is an important step to positively regulate the kinase activity of TESK1.

Localization and functional requirement of yeast Na+/H+ exchanger, Nhx1p, in the endocytic and protein recycling pathway.

Acidification of the lumen of intracellular organelles is important for post-transcriptional processing, endosomal maturation, receptor recycling, and vesicle trafficking, being regulated by an intricate balance between H+ influx through vacuolar-type H+-ATPase and efflux through ion channels and transporters, such as the Na+/H+ exchanger (NHE). The eukaryotic NHE family comprises two major subgroups, one residing in the plasma membrane and the other in intracellular organelles. While mammalian intracellular NHE isoforms are localized to various organelles, including the mid-trans-Golgi compartments, early and late endosomes, and recycling endosomes, Nhx1p, the sole NHE in yeast, has been reported to be localized predominantly to the late endosomal/prevacuolar compartment. Here, using live cell imaging, we demonstrated that Nhx1p is localized to the trans-Golgi network compartments, late endosomes, and recycling endosomes, similar to mammalian intracellular NHE isoforms. Loss of Nhx1p led to accumulation of components of the retromer and endosomal sorting complex required for transport complexes, but not trans-Golgi compartments, in aberrant prevacuolar compartments. Importantly, Nhx1p was also required for recycling of the plasma membrane vesicle SNAP receptor Snc1p. These observations suggest that Nhx1p plays an important role in regulation of the luminal pH of various intracellular organelles, and that this regulation is critical for the protein recycling pathway as well as the endocytic pathway.

Regulation of clathrin coat assembly by Eps15 homology domain–mediated interactions during endocytosis

ETOC: The EH domain is a highly conserved protein–protein interaction domain involved in endocytosis. The EH domains of yeast endocytic proteins, Pan1p, End3p, and Ede1p, have a redundant function and are required for efficient recruitment of several endocytic proteins to sites of endocytosis in order to facilitate clathrin coat assembly.

V-ATPase-dependent luminal acidification is required for endocytic recycling of a yeast cell wall stress sensor, Wsc1p

Wsc1p is a major cell wall sensor protein localized at the polarized cell surface. The localization of Wsc1p is maintained by endocytosis and recycling from endosomes back to the cell surface, but changes to the vacuole when cells are subjected to heat stress. Exploiting this unique property of Wsc1p, we screened for yeast single-gene deletion mutants exhibiting defects in Wsc1p trafficking. By expressing 3GFP-tagged Wsc1p in mutants with deleted genes whose function is related to intracellular trafficking, we identified 5 gene groups affecting Wsc1p trafficking, impaired respectively in endocytic internalization, multivesicular body sorting, the GARP complex, endosomal maturation/vacuolar fusion, and V-ATPase. Interestingly, deletion of the VPH1 gene, encoding the V(o) subunit of vacuolar-type H(+)-ATPase (V-ATPase), led to mis-localization of Wsc1p from the plasma membrane to the vacuole. In addition, disruption of other V-ATPase subunits (vma mutants) also caused defects of Wsc1p trafficking and vacuolar acidification similar to those seen in the vph1Δ mutant. Moreover, we found that deletion of the VPS26 gene, encoding a subunit of the retromer complex, also caused a defect in Wsc1p recycling and mis-localization of Wsc1p to the vacuole. These findings clarified the previously unidentified Wsc1p recycling pathway and requirement of V-ATPase-dependent luminal acidification for Wsc1p recycling.

Bifurcation of the endocytic pathway into Rab5-dependent and -independent transport to the vacuole

The yeast Rab5 homologue, Vps21p, is known to be involved both in the vacuolar protein sorting (VPS) pathway from the trans-Golgi network to the vacuole, and in the endocytic pathway from the plasma membrane to the vacuole. However, the intracellular location at which these two pathways converge remains unclear. In addition, the endocytic pathway is not completely blocked in yeast cells lacking all Rab5 genes, suggesting the existence of an unidentified route that bypasses the Rab5-dependent endocytic pathway. Here we show that convergence of the endocytic and VPS pathways occurs upstream of the requirement for Vps21p in these pathways. We also identify a previously unidentified endocytic pathway mediated by the AP-3 complex. Importantly, the AP-3-mediated pathway appears mostly intact in Rab5-disrupted cells, and thus works as an alternative route to the vacuole/lysosome. We propose that the endocytic traffic branches into two routes to reach the vacuole: a Rab5-dependent VPS pathway and a Rab5-independent AP-3-mediated pathway.

Chemopreventive effects of a low-side-effect antibiotic drug, erythromycin, on mouse intestinal tumors

It is important to establish effective methods for preventing colorectal cancer because the number of colorectal cancer deaths is increasing. Erythromycin one of the macrolide antibiotics, has been shown to exert pleiotropic effects, such as anti-inflammatory and anti-oxidative effects, on mammalian cells. In the present study, we aimed to evaluate the preventive effects of erythromycin on intestinal carcinogenesis. We first confirmed that erythromycin suppresses the transcriptional activity of nuclear factor-κB and activator protein-1 and the expression of its downstream targets, interleukin-6 and cyclooxygenase-2 in human colon cancer cells. Next, we fed 5-week-old male Apc mutant Min mice with diets containing 500 ppm erythromycin for 15 weeks. Erythromycin treatment significantly reduced the number of proximal intestinal polyps to 70.9% of the untreated control value. Moreover, erythromycin reduced the levels of interleukin-6 and cyclooxygenase-2 mRNA expression in intestinal polyps. Although the levels of hepatic NADPH oxidase mRNA were decreased, erythromycin treatment did not affect the levels of oxidative stress markers, reactive carbonyl species, in the liver of Min mice. Our results suggest that erythromycin suppresses intestinal polyp development in Min mice, in part by attenuating local inflammation, and indicate that erythromycin is useful as a chemopreventive agent.

Effects of NSAIDs on the risk factors of colorectal cancer: a mini review

Evidence from epidemiological and experimental studies has shown that non-steroidal anti-inflammatory drugs (NSAIDs) reduce the risk of colorectal cancer (CRC). The function of NSAIDs and the molecular targets for chemopreventive effects on CRC have been extensively studied and their data were reported. However, the relation between NSAIDs and the risk factors of CRC have not been fully elucidated yet. Thus, relations between NSAIDs and the risk factors of CRC, such as overweight and obesity, alcohol, aging, hypertriglyceridemia and smoking, are summarized with our data and with recent reported data in this review.