Caspase 3 exhibits a yeast metacaspase proteostasis function that protects mitochondria from toxic TDP43 aggregates.

Caspase 3 activation is a hallmark of cell death and there is a strong correlation between elevated protease activity and evolving pathology in neurodegenerative disease, such as amyotrophic lateral sclerosis (ALS). At the cellular level, ALS is characterized by protein aggregates and inclusions, comprising the RNA binding protein TDP-43, which are hypothesized to trigger pathogenic activation of caspase 3. However, a growing body of evidence indicates this protease is essential for ensuring cell viability during growth, differentiation and adaptation to stress.

Characterization of angiotensin-converting enzyme 2 ectodomain shedding from mouse proximal tubular cells.

Angiotensin-converting enzyme 2 (ACE2) is highly expressed in the kidney proximal tubule, where it cleaves angiotensin (Ang) II to Ang-(1-7). Urinary ACE2 levels increase in diabetes, suggesting that ACE2 may be shed from tubular cells. The aim of this study was to determine if ACE2 is shed from proximal tubular cells, to characterize ACE2 fragments, and to study pathways for shedding.

The role of Yca1 in proteostasis. Yca1 regulates the composition of the insoluble proteome.

Proteostasis, the process of balancing protein production with protein degradation is vital to normal cell function. Defects within the mechanisms that control proteostasis lead to increased content of a specialized insoluble protein fraction that forms dense aggregates within the cell. We have previously implicated the Saccharomyces cerevisiae metacaspase Yca1 as an active participant in maintaining proteostasis, whereby Yca1 acts to limit aggregate content.

Metacaspase Yca1 is required for clearance of insoluble protein aggregates.

In complex organisms, caspase proteases mediate a variety of cell behaviors, including proliferation, differentiation, and programmed cell death/apoptosis. Structural homologs to the caspase family (termed metacaspases) engage apoptosis in single-cell eukaryotes, yet the molecular mechanisms that contribute to nondeath roles are currently undefined. Here, we report an unexpected role for the Saccharomyces cerevisiae metacaspase Yca1 in protein quality control.

Reconstructing regulatory kinase pathways from phosphopeptide data: a bioinformatics approach.

Protein phosphorylation is a widespread cellular process, and simplistic linear pathway models of kinase signaling likely under-represent the complexity of in vivo pathways. The recent massive increase in information available through protein interaction databases now allows construction of in silico models of protein networks that are underpinned by evidence from real biological systems. By combining protein phosphorylation data with current databases of protein-protein and kinase-substrate interactions, sophisticated models of intracellular protein phosphorylation signaling can be constructed for a system of interest.

A non-death role of the yeast metacaspase: Yca1p alters cell cycle dynamics.

Caspase proteases are a conserved protein family predominantly known for engaging and executing apoptotic cell death. Nevertheless, in higher eukaryotes, caspases also influence a variety of cell behaviors including differentiation, proliferation and growth control. S.

Isolation of phosphoproteins.

Phosphorylation is one of the most abundant post-translational modifications on protein and one that frequently has functional biological consequences. For this reason, screening protein samples for phosphorylations has become an important tool in biochemical research. Affinity purification by immunological or chemical reagents can be used to isolate phosphoproteins from other cellular materials.

Reconstructing the regulatory kinase pathways of myogenesis from phosphopeptide data.

Multiple kinase activities are required for skeletal muscle differentiation. However, the mechanisms by which these kinase pathways converge to coordinate the myogenic process are unknown. Using multiple phosphoprotein and phosphopeptide enrichment techniques we obtained phosphopeptides from growing and differentiating C2C12 muscle cells and determined specific peptide sequences using LC-MS/MS.

A novel PKC regulates ERK activation and degranulation of cytotoxic T lymphocytes: Plasticity in PKC regulation of ERK.

Stimulation of cytotoxic T lymphocyte (CTL) degranulation with plate-bound anti-CD3 Ab leads to two phases of ERK activation: an early PKC-independent phase followed by a later sustained PKC-dependent phase. Herein, we show that a novel PKC (nPKC) mediates the late phase of ERK activation, upstream of Ras in murine T cells. In contrast, when CTL are activated with cross-linked anti-CD3 Ab, which does not trigger CTL degranulation, there is a requirement for conventional PKC (cPKC) for ERK activation.

Identification of candidate regulators of embryonic stem cell differentiation by comparative phosphoprotein affinity profiling.

Embryonic stem cells are a unique cell population capable both of self-renewal and of differentiation into all tissues in the adult organism. Despite the central importance of these cells, little information is available regarding the intracellular signaling pathways that govern self-renewal or early steps in the differentiation program. Embryonic stem cell growth and differentiation correlates with kinase activities, but with the exception of the JAK/STAT3 pathway, the relevant substrates are unknown.

Tyrosine kinase activity and remodelling of the actin cytoskeleton are co-temporally required for degranulation by cytotoxic T lymphocytes.

In this study, we examined the contribution of the actin cytoskeleton to T-cell receptor (TCR)-initiated signalling in cytotoxic T lymphocytes (CTLs). We demonstrate that cytoskeletal remodelling is required for sustaining TCR-stimulated signals that lead to degranulation by CTLs. Disruption of the actin cytoskeleton in CTLs already undergoing signalling responses results in an almost immediate loss of essentially all protein tyrosine phosphorylation.

Phosphatidylinositol-3-kinase regulates PKCtheta activity in cytotoxic T cells.

Protein kinase C (PKC) theta plays a crucial role in T cell activation. We, therefore, examined the regulation of PKCtheta activity in cytotoxic T lymphocytes (CTL). We demonstrated that PMA did not stimulate PKCtheta activation and phospholipase C inhibition did not block anti-CD3-stimulated PKCtheta activation in a CTL clone.

Comparative analysis of phosphoprotein-enriched myocyte proteomes reveals widespread alterations during differentiation.

The differentiation of skeletal muscle has been associated with altered phosphorylation status of individual proteins. However, a global analysis of protein phosphorylation during myogenesis has yet to be undertaken. Here, we report the identification of over 130 putative phosphoproteins from murine C2C12 muscle cells.

Beta 1/beta 3 integrin ligation is uncoupled from ERK1/ERK2 activation in cytotoxic T lymphocytes.

beta 3 integrins mediate fibronectin binding and enhanced activation of cytotoxic T lymphocytes (CTL). The intracellular signals initiated by beta 3 integrins in lymphocytes are not well characterized, but in many cell types, beta 1 integrin ligation activates mitogen-activated protein (MAP) kinases. In the present study, we find that fibronectin can synergize with very low levels of CD3 stimulation to activate the extracellular signal-regulated kinase (ERK)1 and ERK2 MAP kinases but that fibronectin alone induces no detectable MAP kinase activation in CTL.