Yeast Vacuolar HOPS, Regulated by its Kinase, Exploits Affinities for Acidic Lipids and Rab:GTP for Membrane Binding and to Catalyze Tethering and Fusion

Fusion of yeast vacuoles requires the Rab GTPase Ypt7p, four SNAREs (soluble N-ethylmaleimide–sensitive factor attachment protein receptors), the SNARE disassembly chaperones Sec17p/Sec18p, vacuolar lipids, and the Rab-effector complex HOPS (homotypic fusion and vacuole protein sorting). Two HOPS subunits have direct affinity for Ypt7p. Although vacuolar fusion has been reconstituted with purified components, the functional relationships between individual lipids and Ypt7p:GTP have remained unclear. We now report that acidic lipids function with Ypt7p as coreceptors for HOPS, supporting membrane tethering and fusion. After phosphorylation by the vacuolar kinase Yck3p, phospho-HOPS needs both Ypt7p:GTP and acidic lipids to support fusion

Identification of Candidate Casein Kinase 2 Substrates in Mitosis by Quantitative Phosphoproteomics

Protein phosphorylation is a crucial regulatory mechanism that controls many aspects of cellular signaling. Casein kinase 2 (CK2), a constitutively expressed and active kinase, plays key roles in an array of cellular events including transcription and translation, ribosome biogenesis, cell cycle progression, and apoptosis. CK2 is implicated in cancerous transformation and is a therapeutic target in anti-cancer therapy. The specific and selective CK2 ATP competitive inhibitor, CX-4945 (silmitaseratib), is currently in phase 2 clinical trials. While many substrates and interactors of CK2 have been identified, less is known about CK2 substrates in mitosis. In the present work, we utilize CX-4945 and quantitative phosphoproteomics to inhibit CK2 activity in mitotically arrested HeLa cells and determine candidate CK2 substrates. We identify 330 phosphorylation sites on 202 proteins as significantly decreased in abundance upon inhibition of CK2 activity. Motif analysis of decreased sites reveals a linear kinase motif with aspartic and glutamic amino acids downstream of the phosphorylated residues, which is consistent with known substrate preferences for CK2. To validate specific candidate CK2 substrates, we perform in vitro kinase assays using purified components. Furthermore, we identified CK2 interacting proteins by affinity purification-mass spectrometry (AP-MS). To investigate the biological processes regulated by CK2 in mitosis, we perform network analysis and identify an enrichment of proteins involved in chromosome condensation, chromatin organization, and RNA processing. We demonstrate that overexpression of CK2 in HeLa cells affects proper chromosome condensation. Previously, we found that phosphoprotein phosphatase 6 (PP6), but not phosphoprotein phosphatase 2A (PP2A), opposes CK2 phosphorylation of the condensin I complex, which is essential for chromosome condensation. Here, we extend this observation and demonstrate that PP6 opposition of CK2 is a more general cellular regulatory mechanism

The SAURON Project – XIV. No escape from Vesc: a global and local parameter in early-type galaxy evolution

‘The definitive version is available at www3.interscience.wiley.com '. Copyright Royal Astronomical Society. DOI: 10.1111/j.1365-2966.2009.15275.xWe present the results of an investigation of the local escape velocity (Vesc) - line strength index relationship for 48 early type galaxies from the SAURON sample, the first such study based on a large sample of galaxies with both detailed integral field observations and extensive dynamical modelling. Values of Vesc are computed using Multi Gaussian Expansion (MGE) photometric fitting and axisymmetric, anisotropic Jeans' dynamical modelling simultaneously on HST and ground-based images. We determine line strengths and escape velocities at multiple radii within each galaxy, allowing an investigation of the correlation within individual galaxies as well as amongst galaxies. We find a tight correlation between Vesc and the line-strength indices. For Mgb we find that this correlation exists not only between different galaxies but also inside individual galaxies - it is both a local and global correlation. The relation within individual galaxies has the same slope and offset as the global relation to a good level of agreement, though there is significant intrinsic scatter in the local gradients. We transform our line strength index measurements to the single stellar population (SSP) equivalent age (t), metallicity ([Z/H]) and enhancement ([$\alpha$/Fe]) and carry out a principal component analysis of our SSP and Vesc data. We find that in this four-dimensional parameter space the galaxies in our sample are to a good approximation confined to a plane, given by $\log \mathrm({V}_{\mathrm{esc}}/500\mathrm{km/s}) = 0.85 \mathrm{[Z/H]} + 0.43 \log (\mathrm{t}/\mathrm{Gyrs})$ - 0.20. It is surprising that it is a combination of age and metallicity that is conserved; this may indicate a 'conspiracy' between age and metallicity or a weakness in the SSP models.Peer reviewe

PP1:Tautomycetin Complex Reveals a Path toward the Development of PP1-Specific Inhibitors

Selective inhibitors for each serine/threonine phosphatase (PPP) are essential to investigate the biological actions of PPPs and to guide drug development. Biologically diverse organisms (e.g., cyanobacteria, dinoflagellates, beetles) produce structurally distinct toxins that are catalytic inhibitors of PPPs. However, most toxins exhibit little selectivity, typically inhibiting multiple family members with similar potencies. Thus, the use of these toxins as chemical tools to study the relationship between individual PPPs and their biological substrates, and how disruptions in these relationships contributes to human disease, is severely limited. Here, we show that tautomycetin (TTN) is highly selective for a single PPP, protein phosphatase 1 (PP1/PPP1C). Our structure of the PP1:TTN complex reveals that PP1 selectivity is defined by a covalent bond between TTN and a PP1-specific cysteine residue, Cys127. Together, these data provide key molecular insights needed for the development of novel probes targeting single PPPs, especially PP1