Distinct functional outputs of PTEN signalling are controlled by dynamic association with ß-arrestins.

The tumour suppressor PTEN (phosphatase and tensin deleted on chromosome 10) regulates major cellular functions via lipid phosphatase-dependent and -independent mechanisms. Despite its fundamental pathophysiological importance, how PTEN's cellular activity is regulated has only been partially elucidated. We report that the scaffolding proteins ß-arrestins (ß-arrs) are important regulators of PTEN. Downstream of receptor-activated RhoA/ROCK signalling, ß-arrs activate the lipid phosphatase activity of PTEN to negatively regulate Akt and cell proliferation. In contrast, following wound-induced RhoA activation, ß-arrs inhibit the lipid phosphatase-independent anti-migratory effects of PTEN. ß-arrs can thus differentially control distinct functional outputs of PTEN important for cell proliferation and migration.

Structure-function analysis of core STRIPAK Proteins: a signaling complex implicated in Golgi polarization.

Cerebral cavernous malformations (CCMs) are alterations in brain capillary architecture that can result in neurological deficits, seizures, or stroke. We recently demonstrated that CCM3, a protein mutated in familial CCMs, resides predominantly within the STRIPAK complex (striatin interacting phosphatase and kinase). Along with CCM3, STRIPAK contains the Ser/Thr phosphatase PP2A. The PP2A holoenzyme consists of a core catalytic subunit along with variable scaffolding and regulatory subunits. Within STRIPAK, striatin family members act as PP2A regulatory subunits. STRIPAK also contains all three members of a subfamily of Sterile 20 kinases called the GCKIII proteins (MST4, STK24, and STK25). Here, we report that striatins and CCM3 bridge the phosphatase and kinase components of STRIPAK and map the interacting regions on each protein. We show that striatins and CCM3 regulate the Golgi localization of MST4 in an opposite manner. Consistent with a previously described function for MST4 and CCM3 in Golgi positioning, depletion of CCM3 or striatins affects Golgi polarization, also in an opposite manner. We propose that STRIPAK regulates the balance between MST4 localization at the Golgi and in the cytosol to control Golgi positioning.

The structure of the human RNase H2 complex defines key interaction interfaces relevant to enzyme function and human disease.

Ribonuclease H2 (RNase H2) is the major nuclear enzyme involved in the degradation of RNA/DNA hybrids and removal of ribonucleotides misincorporated in genomic DNA. Mutations in each of the three RNase H2 subunits have been implicated in a human auto-inflammatory disorder, Aicardi-Goutières Syndrome (AGS). To understand how mutations impact on RNase H2 function we determined the crystal structure of the human heterotrimer. In doing so, we correct several key regions of the previously reported murine RNase H2 atomic model and provide biochemical validation for our structural model. Our results provide new insights into how the subunits are arranged to form an enzymatically active complex. In particular, we establish that the RNASEH2A C terminus is a eukaryotic adaptation for binding the two accessory subunits, with residues within it required for enzymatic activity. This C-terminal extension interacts with the RNASEH2C C terminus and both are necessary to form a stable, enzymatically active heterotrimer. Disease mutations cluster at this interface between all three subunits, destabilizing the complex and/or impairing enzyme activity. Altogether, we locate 25 out of 29 residues mutated in AGS patients, establishing a firm basis for future investigations into disease pathogenesis and function of the RNase H2 enzyme.

A scanning peptide array approach uncovers association sites within the JNK/beta arrestin signalling complex.

Beta arrestins are molecular scaffolds that can bring together three-component mitogen-activated protein kinase signalling modules to promote signal compartmentalisation. We use peptide array technology to define novel interfaces between components within the c-Jun N-terminal kinase (JNK)/beta arrestin signalling complex. We show that beta arrestin 1 and beta arrestin 2 associate with JNK3 via the kinase N-terminal domain in a region that, surprisingly, does not harbour a known 'common docking' motif. In the N-domain and C-terminus of beta arrestin 1 and beta arrestin 2 we identify two novel apoptosis signal-regulating kinase 1 binding sites and in the N-domain of the beta arrestin 1 and beta arrestin 2 we identify a novel MKK4 docking site.

The inhibitory profile of Ibudilast against the human phosphodiesterase enzyme family.

Ibudilast is widely used in Japan to treat ischemic stroke and bronchial asthma. Its mode of action is through the inhibition of cyclic nucleotide phosphodiesterases (PDEs). Growing evidence suggests this compound has utility in a range of neurological conditions linked to its ability to elevate cellular cyclic nucleotide concentrations, however limited data exists on Ibudilast's action on individual PDE families. We therefore used an extensive panel of human PDE enzymes to define the PDE inhibitory profile of this compound. Ibudilast preferentially inhibits PDE3A, PDE4, PDE10 and PDE11 with lesser inhibition of a number of other families. The significance of these findings is discussed in relation to Ibudilast's observed effects on certain disease states.

Evidence that there are two copies of subunit c" in V0 complexes in the vacuolar H+-ATPase.

The proton-translocating core of eukaryotic vacuolar H(+)-ATPase (V-ATPase), V(0) consists of a hexameric arrangement of transmembrane alpha-helices formed from the related polypeptides, subunit c and subunit c". The former is comprised of four transmembrane alpha-helices, whilst the latter has an extra transmembrane domain at its N-terminus. In addition, the fungal form of V(0) contains a minor subunit c-related polypeptide, subunit c'. All three are required for activity of the proton pump in Saccharomyces cerevisiae. We have introduced cysteine residues in the N-terminal extension of subunit c" in a cysteine-free form. All mutant forms are active in the V-ATPase from S. cerevisiae. Oxidation of vacuolar membranes containing the cysteine-replaced forms gave a cross-linked product of 42000Da. Analysis of this species showed it to be a dimeric form of subunit c", and further studies confirmed there are two copies of subunit c" in the V-ATPases in which it is present. Co-expression of double cysteine-replaced forms of both subunit c and c" gave rise to only homotypic cross-linked forms. Also, subunit c oligomeric complexes are present in vacuolar membranes in the absence of subunit c", consistent with previous observations showing hexameric arrangements of subunit c in gap-junction-like membranes. In vitro studies showed subunit c" can bind to subunit c and itself. The extent of binding can be increased by removal of the N-terminal domain of subunit c". This domain may therefore function to limit the copy number of subunit c" in V(0). A deletion study shows that the domain is essential for the activity of subunit c". The results can be combined into a model of V(0) which contains two subunit c" protomers with the extra transmembrane domain located toward the central pore. Thus the predicted stoichiometry of V(0) in which subunit c" is present is subunit c(3):subunit c'(1):subunit c"(2). On the basis of the mutational and binding studies, it seems likely that two copies of subunit c" are next to each other.