Akira Kakizuka

The small GTP-binding protein Rho binds to and activates a 160 kDa Ser/Thr protein kinase homologous to myotonic dystrophy kinase.

The small GTP‐binding protein Rho functions as a molecular switch in the formation of focal adhesions and stress fibers, cytokinesis and transcriptional activation. The biochemical mechanism underlying these actions remains unknown. Using a ligand overlay assay, we purified a 160 kDa platelet protein that bound specifically to GTP‐bound Rho. This protein, p160, underwent autophosphorylation at its serine and threonine residues and showed the kinase activity to exogenous substrates. Both activities were enhanced by the addition of GTP‐bound Rho. A cDNA encoding p160 coded for a 1354 amino acid protein. This protein has a Ser/Thr kinase domain in its N‐terminus, followed by a coiled‐coil structure approximately 600 amino acids long, and a cysteine‐rich zinc finger‐like motif and a pleckstrin homology region in the C‐terminus. The N‐terminus region including a kinase domain and a part of coiled‐coil structure showed strong homology to myotonic dystrophy kinase over 500 residues. When co‐expressed with RhoA in COS cells, p160 was co‐precipitated with the expressed Rho and its kinase activity was activated, indicating that p160 can associate physically and functionally with Rho both in vitro and in vivo.

p140mDia, a mammalian homolog of Drosophila diaphanous, is a target protein for Rho small GTPase and is a ligand for profilin

Rho small GTPase regulates cell morphology, adhesion and cytokinesis through the actin cytoskeleton. We have identified a protein, p140mDia, as a downstream effector of Rho. It is a mammalian homolog of Drosophila diaphanous, a protein required for cytokinesis, and belongs to a family of formin‐related proteins containing repetitive polyproline stretches. p140mDia binds selectively to the GTP‐bound form of Rho and also binds to profilin. p140mDia, profilin and RhoA are co‐localized in the spreading lamellae of cultured fibroblasts. They are also co‐localized in membrane ruffles of phorbol ester‐stimulated sMDCK2 cells, which extend these structures in a Rho‐dependent manner. The three proteins are recruited around phagocytic cups induced by fibronectin‐coated beads. Their recruitment is not induced after Rho is inactivated by microinjection of botulinum C3 exoenzyme. Overexpression of p140mDia in COS‐7 cells induced homogeneous actin filament formation. These results suggest that Rho regulates actin polymerization by targeting profilin via p140mDia beneath the specific plasma membranes.

Caspase Cleavage of Gene Products Associated with Triplet Expansion Disorders Generates Truncated Fragments Containing the Polyglutamine Tract

The neurodegenerative diseases Huntington disease, dentatorubropallidoluysian atrophy, spinocerebellar atrophy type 3, and spinal bulbar muscular atrophy are caused by expansion of a polyglutamine tract within their respective gene products. There is increasing evidence that generation of truncated proteins containing an expanded polyglutamine tract may be a key step in the pathogenesis of these disorders. We now report that, similar to huntingtin, atrophin-1, ataxin-3, and the androgen receptor are cleaved in apoptotic extracts. Furthermore, each of these proteins is cleaved by one or more purified caspases, cysteine proteases involved in apoptotic death. The CAG length does not modulate susceptibility to cleavage of any of the full-length proteins. Our results suggest that by generation of truncated polyglutamine-containing proteins, caspase cleavage may represent a common step in the pathogenesis of each of these neurodegenerative diseases.

Expanded polyglutamine in the Machado–Joseph disease protein induces cell death in vitro and in vivo

Recently, we identified a novel gene, MJD1, which contains an expanded GAG triplet repeat in Machado–Joseph disease. Here we report the induction of apoptosis in cultured cells expressing a portion of the MJD1 gene that includes the expanded GAG repeats. Cell death occurs only when the GAG repeat is translated into polyglutamine residues, which apparently precipitate in large covalently modified forms. We also created ataxic transgenic mice by expressing the expanded polyglutamine stretch in Purkinje cells. Our results demonstrate the potential involvement of the expanded polyglutamine as the common aetiological agent for inherited neurodegenerative diseases with GAG expansions.

Protein kinase N (PKN) and PKN-related protein rhophilin as targets of small GTPase Rho

The Rho guanosine 5′-triphosphatase (GTPase) cycles between the active guanosine triphosphate (GTP)-bound form and the inactive guanosine diphosphate-bound form and regulates cell adhesion and cytokinesis, but how it exerts these actions is unknown. The yeast two-hybrid system was used to clone a complementary DNA for a protein (designated Rhophilin) that specifically bound to GTP-Rho. The Rho-binding domain of this protein has 40 percent identity with a putative regulatory domain of a protein kinase, PKN. PKN itself bound to GTP-Rho and was activated by this binding both in vitro and in vivo. This study indicates that a serine-threonine protein kinase is a Rho effector and presents an amino acid sequence motif for binding to GTP-Rho that may be shared by a family of Rho target proteins.

PPARγ coactivator 1β/ERR ligand 1 is an ERR protein ligand, whose expression induces a high-energy expenditure and antagonizes obesity

A well balanced body energy budget controlled by limitation of calorie uptake and/or increment of energy expenditure, which is typically achieved by proper physical exercise, is most effective against obesity and diabetes mellitus. Recently, peroxisome proliferator-activated receptor (PPAR) γ, a member of the nuclear receptor, and its cofactors have been shown to be involved in lipid metabolism and in the control of energy expenditure. Here we show that PPARγ coactivator 1 (PGC-1) β functions as ERRL1 (for ERR ligand 1), which can bind and activate orphan ERRs (estrogen receptor-related receptors) in vitro. Consistently, PGC-1β/ERRL1 transgenic mice exhibit increased expression of the medium-chain acyl CoA dehydrogenase, a known ERR target and a pivotal enzyme of mitochondrial β-oxidation in skeletal muscle. As a result, the PGC-1β/ERRL1 mice show a state similar to an athlete; namely, the mice are hyperphagic and of elevated energy expenditure and are resistant to obesity induced by a high-fat diet or by a genetic abnormality. These results demonstrate that PGC-1β/ERRL1 can function as a protein ligand of ERR, and that its level contributes to the control of energy balance in vivo, and provide a strategy for developing novel antiobesity drugs.

Listeria monocytogenes ActA-mediated escape from autophagic recognition

Autophagy degrades unnecessary organelles and misfolded protein aggregates, as well as cytoplasm-invading bacteria. Nevertheless, the bacteria Listeria monocytogenes efficiently escapes autophagy. We show here that recruitment of the Arp2/3 complex and Ena/VASP, via the bacterial ActA protein, to the bacterial surface disguises the bacteria from autophagic recognition, an activity that is independent of the ability to mediate bacterial motility. L. monocytogenes expressing ActA mutants that lack the ability to recruit the host proteins initially underwent ubiquitylation, followed by recruitment of p62 (also known as SQSTM1) and LC3, before finally undergoing autophagy. The ability of ActA to mediate protection from ubiquitylation was further demonstrated by generating aggregate-prone GFP–ActA–Q79C and GFP–ActA–170* chimaeras, consisting of GFP (green fluorescent protein), the ActA protein and segments of polyQ or Golgi membrane protein GCP170 (ref. 6). GFP–ActA–Q79C and GFP–ActA–170* formed aggregates in the host cell cytoplasm, however, these ActA-containing aggregates were not targeted for association with ubiquitin and p62. Our findings indicate that ActA-mediated host protein recruitment is a unique bacterial disguise tactic to escape from autophagy.

Two thromboxane A2 receptor isoforms in human platelets: Opposite coupling to adenylyl cyclase with different sensitivity to Arg60 to Leu mutation

Thromboxane A2 (TXA2) receptor is a key molecule in hemostasis as its abnormality leads to bleeding disorders. Two isoforms of the human TXA2 receptor have been cloned; one from placenta and the other from endothelium, here referred to as TXR alpha and TXR beta, respectively. These isoforms differ only in their carboxyl-terminal tails. We report that both isoforms are present in human platelets. The two isoforms expressed in cultured cells show similar ligand binding characteristics and phospholipase C (PLC) activation but oppositely regulate adenylyl cyclase activity; TXR alpha activates adenylyl cyclase, while TXR beta inhibits it. The Arg60 to Leu mutant of TXR alpha, which has been shown to impair PLC activation (Hirata, T., A. Kakizuka, F. Ushikubi, I. Fuse, M. Okuma, and S. Narumiya. 1994. J. Clin. Invest. 94: 1662-1667), also impairs adenylyl cyclase stimulation, whereas that of TXR beta retains its activity to inhibit adenylyl cyclase. These findings suggest that the pathway linked to adenylyl cyclase inhibition might be involved in some of the TXA2-induced platelet responses such as shape change and phospholipase A2 activation which remain unaffected in the patients with this mutation.

VCP/p97 in abnormal protein aggregates, cytoplasmic vacuoles, and cell death, phenotypes relevant to neurodegeneration.

Neuronal cell death, abnormal protein aggregates, and cytoplasmic vacuolization are major pathologies observed in many neurodegenerative disorders such as the polyglutamine (polyQ) diseases, prion disease, Alzheimer disease, and the Lewy body diseases, suggesting common mechanisms underlying neurodegeneration. Here, we have identified VCP/p97, a member of the AAA+ family of ATPase proteins, as a polyQ-interacting protein in vitro and in vivo, and report on its characterization. Endogenous VCP co-localized with expanded polyQ (ex-polyQ) aggregates in cultured cells expressing ex-polyQ, with nuclear inclusions in Huntington disease patient brains, and with Lewy bodies in patient samples. Moreover, the expression of VCP mutants with mutations in the 2nd ATP binding domain created cytoplasmic vacuoles, followed by cell death. Very similar vacuoles were also induced by ex-polyQ expression or proteasome inhibitor treatment. These results suggest that VCP functions not only as a recognition factor for abnormally folded proteins but also as a pathological effector for several neurodegenerative phenotypes. VCP may thus be an ideal molecular target for the treatment of neurodegenerative disorders. Cell Death and Differentiation (2001) 8, 977–984

α,β-Unsaturated Ketone Is a Core Moiety of Natural Ligands for Covalent Binding to Peroxisome Proliferator-activated Receptor γ

Peroxisome proliferator-activated receptor γ (PPARγ) functions in various biological processes, including macrophage and adipocyte differentiation. Several natural lipid metabolites have been shown to activate PPARγ. Here, we report that some PPARγ ligands, including 15-deoxy-Δ12,14-prostaglandin J2, covalently bind to a cysteine residue in the PPARγ ligand binding pocket through a Michael addition reaction by an α,β-unsaturated ketone. Using rhodamine-maleimide as well as mass spectroscopy, we showed that the binding of these ligands is covalent and irreversible. Consistently, mutation at the cysteine residue abolished abilities of these ligands to activate PPARγ, but not of BRL49653, a non-covalent synthetic agonist, indicating that covalent binding of the α,β-unsaturated ketone in the natural ligands was required for their transcriptional activities. Screening of lipid metabolites containing the α,β-unsaturated ketone revealed that several other oxidized metabolites of hydroxyeicosatetraenoic acid, hydroxyeicosadecaenoic acid, and prostaglandins can also function as novel covalent ligands for PPARγ. We propose that PPARγ senses oxidation of fatty acids by recognizing such an α,β-unsaturated ketone as a common moiety.