Masato Umikawa

A downstream target of RHO1 small GTP-binding protein is PKC1, a homolog of protein kinase C, which leads to activation of the MAP kinase cascade in Saccharomyces cerevisiae.

The RHO1 gene in Saccharomyces cerevisiae encodes a homolog of the mammalian RhoA small GTP‐binding protein, which is implicated in various actin cytoskeleton‐dependent cell functions. In yeast, Rho1p is involved in bud formation. A yeast strain in which RHO1 is replaced with RhoA shows a recessive temperature‐sensitive growth phenotype. A dominant suppressor mutant was isolated from this strain. Molecular cloning of the suppressor gene revealed that the mutation occurred at the pseuodosubstrate site of PKC1, a yeast homolog of mammalian protein kinase C. Two‐hybrid analysis demonstrated that GTP‐Rho1p, but not GDP‐Rho1p, interacted with the region of Pkc1p containing the pseudosubstrate site and the C1 domain. MKK1 and MPK1 encode MAP kinase kinase and MAP kinase homologs, respectively, and function downstream of PKC1. A dominant active MKK1–6 mutation or overexpression of MPK1 suppressed the temperature sensitivity of the RhoA mutant. The dominant activating mutation of PKC1 suppressed the temperature sensitivity of the RhoA mutant. The dominant activating mutation of PKC1 suppressed the temperature sensitivity of two effector mutants of RHO1, rho1(F44Y) and rho1(E451), but not that of rho1(V43T). These results indicate that there are at least two signaling pathways regulated by Rho1p and that one of the downstream targets is Pkc1p, leading to the activation of the MAP kinase cascade.

Bni1p and Bnr1p: downstream targets of the Rho family small G‐proteins which interact with profilin and regulate actin cytoskeleton in Saccharomyces cerevisiae

The RHO1 gene encodes a homologue of mammalian RhoA small G‐protein in the yeast Saccharomyces cerevisiae. Rho1p is required for bud formation and is localized at a bud tip or a cytokinesis site. We have recently shown that Bni1p is a potential target of Rho1p. Bni1p shares the FH1 and FH2 domains with proteins involved in cytokinesis or establishment of cell polarity. In S.cerevisiae, there is an open reading frame (YIL159W) which encodes another protein having the FH1 and FH2 domains and we have named this gene BNR1 (BNI1 Related). Bnr1p interacts with another Rho family member, Rho4p, but not with Rho1p. Disruption of BNI1 or BNR1 does not show any deleterious effect on cell growth, but the bni1 bnr1 mutant shows a severe temperature‐sensitive growth phenotype. Cells of the bni1 bnr1 mutant arrested at the restrictive temperature are deficient in bud emergence, exhibit a random distribution of cortical actin patches and often become multinucleate. These phenotypes are similar to those of the mutant of PFY1, which encodes profilin, an actin‐binding protein. Moreover, yeast two‐hybrid and biochemical studies demonstrate that Bni1p and Bnr1p interact directly with profilin at the FH1 domains. These results indicate that Bni1p and Bnr1p are potential targets of the Rho family members, interact with profilin and regulate the reorganization of actin cytoskeleton.

Bni1p implicated in cytoskeletal control is a putative target of Rho1p small GTP binding protein in Saccharomyces cerevisiae.

The RHO1 gene encodes a homolog of mammalian RhoA small GTP binding protein in the yeast Saccharomyces cerevisiae. Rho1p is localized at the growth sites, including the bud tip and the cytokinesis site, and is required for bud formation. We have recently shown that Pkc1p, a yeast homolog of mammalian protein kinase C, and glucan synthase are targets of Rho1p. Using the two‐hybrid screening system, we cloned a gene encoding a protein which interacted with the GTP‐bound form of Rho1p. This gene was identified as BNI1, known to be implicated in cytokinesis or establishment of cell polarity in S.cerevisiae. Bni1p shares homologous domains (FH1 and FH2 domains) with proteins involved in cytokinesis or establishment of cell polarity, including formin of mouse, capu and dia of Drosophila and FigA of Aspergillus. A temperature‐sensitive mutation in which the RHO1 gene was replaced by the mammalian RhoA gene showed a synthetically lethal interaction with the bni1 mutation and the RhoA bni1 mutant accumulated cells with a deficiency in cytokinesis. Furthermore, this synthetic lethality was caused by the incapability of RhoA to activate Pkc1p, but not glucan synthase. These results suggest that Rho1p regulates cytoskeletal reorganization at least through Bni1p and Pkc1p.

Interaction of Bnr1p with a Novel Src Homology 3 Domain-containing Hof1p IMPLICATION IN CYTOKINESIS IN SACCHAROMYCES CEREVISIAE

Proteins containing the formin homology (FH) domains FH1 and FH2 are involved in cytokinesis or establishment of cell polarity in a variety of organisms. We have shown that the FH proteins Bni1p and Bnr1p are potential targets of the Rho family small GTP-binding proteins and bind to an actin-binding protein, profilin, at their proline-rich FH1 domains to regulate reorganization of the actin cytoskeleton in the yeast Saccharomyces cerevisiae. We found here that a novel Src homology 3 (SH3) domain-containing protein, encoded by YMR032w, interacted with Bnr1p in a GTP-Rho4p-dependent manner through the FH1 domain of Bnr1p and the SH3 domain of Ymr032wp. Ymr032wp weakly bound to Bni1p. Ymr032wp was homologous to cdc15p, which is involved in cytokinesis inSchizosaccharomyces pombe, and we named this geneHOF1 (homolog of cdc 15). Both Bnr1p and Hof1p were localized at the bud neck, and both the bnr1 andhof1 mutations showed synthetic lethal interactions with the bni1 mutation. The hof1 mutant cells showed phenotypes similar to those of the septin mutants, indicating thatHOF1 is involved in cytokinesis. These results indicate that Bnr1p directly interacts with Hof1p as well as with profilin to regulate cytoskeletal functions in S. cerevisiae.

Interaction of Rho1p target Bni1p with F-actin-binding elongation factor 1α : implication in Rho1p-regulated reorganization of the actin cytoskeleton in Saccharomyces cerevisiae

The RHO1 gene encodes a homolog of mammalian RhoA small G protein in the yeast Saccharomyces cerevisiae. We have shown that Bni1p is one of the downstream targets of Rho1p and regulates reorganization of the actin cytoskeleton through the interaction with profilin, an actin monomer-binding protein. A Bni1p-binding protein was affinity purified from the yeast cytosol fraction and was identified to be Tef1p/Tef2p, translation elongation factor 1α (EF1α). EF1α is an essential component of the protein synthetic machinery and also possesses the actin filament (F-actin)-binding and -bundling activities. EF1α bound to the 186 amino acids region of Bni1p, located between the FH1 domain, the proline-rich profilin-binding domain, and the FH2 domain, of which function is not known. The binding of Bni1p to EF1α inhibited its F-actin-binding and -bundling activities. The BNI1 gene deleted in the EF1α-binding region did not suppress the bni1 bnr1 mutation in which the actin organization was impaired. These results suggest that the Rho1p–Bni1p system regulates reorganization of the actin cytoskeleton through the interaction with both EF1α and profilin.

Association of frabin with the actin cytoskeleton is essential for microspike formation through activation of Cdc42 small G protein.

We have recently isolated a novel actin filament-binding protein, named frabin. Frabin has one actin filament-binding domain (ABD), one Dbl homology domain (DHD), first pleckstrin homology domains (PHD) adjacent to DHD, one cysteine rich-domain (CRD), and second PHD from the N terminus to the C terminus in this order. Full-length frabin induces microspike formation and c-Jun N-terminal kinase (JNK) activation. We found here that the fragment of frabin containing DHD and first PHD stimulated guanine nucleotide exchange of Cdc42Hs small G protein, but not that of RhoA or Rac1 small G protein. However, this fragment of frabin did not induce microspike formation, and ABD was additionally necessary for microspike formation. Frabin having ABD was associated with the actin cytoskeleton, whereas frabin lacking ABD was diffusely distributed in the cytoplasm. In contrast, ABD was not necessary for JNK activation but CRD and second PHD were additionally necessary for this activation. These results indicate that the association of frabin with the actin cytoskeleton is essential for microspike formation but not for JNK activation and that different domains of frabin are involved in microspike formation and JNK activation through Cdc42 activation.

MINK is a Rap2 effector for phosphorylation of the postsynaptic scaffold protein TANC1.

Rap1 and Rap2 are similar Ras-like G proteins but perform distinct functions. By the affinity chromatography/mass-spectrometry approach and the yeast two-hybrid screening, we identified Misshapen/NIKs-related kinase (MINK) as a novel Rap2-interacting protein that does not interact with Rap1 or Ras. MINK is a member of the STE20 group of mitogen-activated protein kinase kinase kinase kinases. The interaction between MINK and Rap2 was GTP-dependent and required Phe39 within the effector region of Rap2; the corresponding residue in Rap1 and Ras is Ser. MINK was enriched in the brain, and both MINK and its close relative, Traf2- and Nck-interacting kinase (TNIK), interacted with a postsynaptic scaffold protein containing tetratricopeptide repeats, ankyrin repeats and a coiled-coil region (TANC1) and induced its phosphorylation, under control of Rap2 in cultured cells. These are novel actions of MINK and TNIK, and consistent with a role of MINK as a Rap2 effector in the brain.

Ectopic coexpression of keratin 8 and 18 promotes invasion of transformed keratinocytes and is induced in patients with cutaneous squamous cell carcinoma

Cutaneous squamous cell carcinoma (cSCC) results from transformation of epidermal keratinocytes. Invasion of transformed keratinocytes through the basement membrane into the dermis results in invasive cSCC with substantial metastatic potential. To better understand the mechanisms for invasion and metastasis, we compared the protein expression profiles of a non-metastatic transformed mouse keratinocyte line and its metastatic derivative. Keratin 8 (Krt8) and Krt18, not seen in normal keratinocytes, were coexpressed and formed Krt8/18 filaments in the metastatic line. The metastatic line efficiently invaded an artificial basement membrane in vitro owing to the Krt8/18-coexpression, since coexpression of exogenous Krt8/18 in the non-invasive parental line conferred invasiveness. To test whether the Krt8/18-coexpression is induced and is involved in cSCC invasion, we examined specimens from 21 pre-invasive and 24 invasive cSCC patients by immunohistochemistry, and the ectopic Krt8/18-coexpression was almost exclusively found in invasive cSCC. Further studies are needed to examine the clinical significance of ectopic Krt8/18-coexpression in cSCC.

Rap2 function requires palmitoylation and recycling endosome localization

Rap2A, Rap2B, and Rap2C are Ras-like small G proteins. The role of their post-translational processing has not been investigated due to the lack of information on their downstream signaling. We have recently identified the Traf2- and Nck-interacting kinase (TNIK), a member of the STE20 group of mitogen-activated protein kinase kinase kinase kinases, as a specific Rap2 effector. Here we report that, in HEK293T cells, Rap2A (farnesylated) and Rap2C (likely farnesylated), but not Rap2B (geranylgeranylated), require palmitoylation for membrane-association and TNIK activation, whereas all Rap2 proteins, including Rap2B, require palmitoylation for induction of TNIK-mediated phenotype, the suppression of cell spreading. Furthermore, we report for the first time that, in COS-1 cells, Rap2 proteins localize, and recruit TNIK, to the recycling endosomes, but not the Golgi nor the endoplasmic reticulum, in a palmitoylation-dependent manner. These observations implicate the involvement of palmitoylation and recycling endosome localization in cellular functions of Rap2 proteins.

Proteomic Analysis of the Trabecular Meshwork of Rats in a Steroid-Induced Ocular Hypertension Model: Downregulation of Type I Collagen C-Propeptides

Purpose: To investigate global protein expression profiles in the trabecular meshwork (TM) of normal and glucocorticoid-induced ocular hypertensive rat eyes by proteomic analysis, which has not yet been conducted to date. Materials and Methods: A rat ocular hypertension model was produced by topical application of dexamethasone (DEX) for 4 weeks. Age-matched untreated rats served as controls. Intraocular pressure (IOP) was monitored by an electronic tonometer. TM protein expression profiling and protein identification was carried out by a two-dimensional fluorescence differential gel electrophoresis (2-D DIGE) system and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, respectively. Results: In DEX-treated rats, average IOP was elevated significantly, as compared with controls. By the DEX treatment, 14 TM protein spots were up- or downregulated consistently in 2-D DIGE analyses. Proteins exhibiting more than 2-fold statistically significant change were identified by MALDI-TOF mass spectrometry. αA-Crystallin and βA3-crystallin were upregulated, while the C-propeptides of type I collagen were downregulated. Conclusion: Relatively short-term glucocorticoid application induced alteration in the expression of a number of proteins, including downregulation of type I collagen C-propeptides. This could reflect impaired collagen turnover in the TM of glucocorticoid-treated eyes.