DRAK2 regulates myosin light chain phosphorylation in T cells.

Death-associated protein kinase-related apoptosis-inducing kinase-2 (DRAK2; also known as STK17B) is a serine/threonine kinase expressed in T cells. Drak2-deficient (Drak2-/-) mice respond effectively to tumors and pathogens while displaying resistance to T cell-mediated autoimmune disease. However, the molecular mechanisms by which DRAK2 impacts T cell function remain unclear.

Mitophagy restricts BAX/BAK-independent, Parkin-mediated apoptosis.

Degradation of defective mitochondria is an essential process to maintain cellular homeostasis and it is strictly regulated by the ubiquitin-proteasome system (UPS) and lysosomal activities. Here, using genome-wide CRISPR and small interference RNA screens, we identified a critical contribution of the lysosomal system in controlling aberrant induction of apoptosis following mitochondrial damage. After treatment with mitochondrial toxins, activation of the PINK1-Parkin axis triggered a BAX- and BAK-independent process of cytochrome c release from mitochondria followed by APAF1 and caspase 9-dependent apoptosis.

Ca-mediated mitochondrial inner membrane permeabilization induces cell death independently of Bax and Bak.

The ability of mitochondria to buffer a rapid rise in cytosolic Ca is a hallmark of proper cell homeostasis. Here, we employed m-3M3FBS, a putative phospholipase C (PLC) agonist, to explore the relationships between intracellular Ca imbalance, mitochondrial physiology, and cell death. m-3M3FBS induced a potent dose-dependent Ca release from the endoplasmic reticulum (ER), followed by a rise in intra-mitochondrial Ca.

Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis.

Necroptosis is a programmed cell death pathway triggered by activation of receptor interacting protein kinase 3 (RIPK3), which phosphorylates and activates the mixed lineage kinase-like domain pseudokinase, MLKL, to rupture or permeabilize the plasma membrane. Necroptosis is an inflammatory pathway associated with multiple pathologies including autoimmunity, infectious and cardiovascular diseases, stroke, neurodegeneration, and cancer. Here, we describe protocols that can be used to characterize MLKL as the executioner of plasma membrane rupture in necroptosis.

Metabolic maintenance of cell asymmetry following division in activated T lymphocytes.

Asymmetric cell division, the partitioning of cellular components in response to polarizing cues during mitosis, has roles in differentiation and development. It is important for the self-renewal of fertilized zygotes in Caenorhabditis elegans and neuroblasts in Drosophila, and in the development of mammalian nervous and digestive systems. T lymphocytes, upon activation by antigen-presenting cells (APCs), can undergo asymmetric cell division, wherein the daughter cell proximal to the APC is more likely to differentiate into an effector-like T cell and the distal daughter is more likely to differentiate into a memory-like T cell.

Sequential Engagement of Distinct MLKL Phosphatidylinositol-Binding Sites Executes Necroptosis.

Necroptosis is a cell death pathway regulated by the receptor interacting protein kinase 3 (RIPK3) and the mixed lineage kinase domain-like (MLKL) pseudokinase. How MLKL executes plasma membrane rupture upon phosphorylation by RIPK3 remains controversial. Here, we characterize the hierarchical transduction of structural changes in MLKL that culminate in necroptosis.

Analysis of nondegradative protein ubiquitylation with a monoclonal antibody specific for lysine-63-linked polyubiquitin.

Modification of proteins by the addition of lysine (K)-63-linked polyubiquitin (polyUb) chains is suggested to play important roles in a variety of cellular events, including DNA repair, signal transduction, and receptor endocytosis. However, identifying such modifications in living cells is complex and cumbersome. We have generated a monoclonal antibody (mAb) that specifically recognizes K63-linked polyUb, but not any other isopeptide-linked (K6, K11, K27, K29, K33, or K48) polyUb or monoubiquitin.