Purification of B-50 by 2-mercaptoethanol extraction from rat brain synaptosomal plasma membranes

Several methods have been described previously for the purification of the nervous-tissue specific protein kinase C substrate B-50 (GAP-43). In this paper we present a new purification method for B-50 from rat brain which employs 2-mercaptoethanol to release the protein from isolated synaptosomal plasma membranes. Most likely, 2-mercaptoethanol reduces disulfide bonds involved in the linkage of B-50 to the membrane. After washing the membranes with 100 mM NaCl to detach loosely bound proteins, B-50 is the major protein (and the only protein kinase C substrate) released by 0.5% 2-mercaptoethanol treatment. Further purification to apparent homogeneity is achieved by affinity chromatography on calmodulin sepharose. B-50 binds to calmodulin in the absence of calcium and specifically elutes from the column with 3 mM calcium. The procedures described is simple, rapid and highly suitable for large scale purification of B-50 from rat brain.     

Structural Insights into the Mechanism of Negative Regulation of Single-box High Mobility Group Proteins by the Acidic Tail Domain

The Drosophila and plant (maize) functional counterparts of the abundant vertebrate chromosomal protein HMGB1 (HMG-D and ZmHMGB1, respectively) differ from HMGB1 in having a single HMG box, as well as basic and acidic flanking regions that vary greatly in length and charge. We show that despite these variations, HMG-D and ZmHMGB1 exist in dynamic assemblies in which the basic HMG boxes and linkers associate with their intrinsically disordered, predominantly acidic, tails in a manner analogous to that observed previously for HMGB1. The DNA-binding surfaces of the boxes and linkers are occluded in “auto-inhibited” forms of the protein, which are in equilibrium with transient, more open structures that are “binding-competent.” This strongly suggests that the mechanism of auto-inhibition may be a general one. HMG-D and ZmHMGB1 differ from HMGB1 in having phosphorylation sites in their tail and linker regions. In both cases, in vitro phosphorylation of serine residues within the acidic tail stabilizes the assembled form, suggesting another level of regulation for interaction with DNA, chromatin, and other proteins that is not possible for the uniformly acidic (hence unphosphorylatable) tail of HMGB1.     

Scaffold State Switching Amplifies,Accelerates, and Insulates Protein Kinase C Signaling

Scaffold proteins localize two or more signaling enzymes in close proximity to their downstream effectors. A-kinase-anchoring proteins (AKAPs) are a canonical family of scaffold proteins known to bind protein kinase A (PKA) and other enzymes. Several AKAPs have been shown to accelerate, amplify, and specify signal transduction to dynamically regulate numerous cellular processes. However, there is little theory available to mechanistically explain how signaling on protein scaffolds differs from solution biochemistry. In our present study, we propose a novel kinetic mechanism for enzymatic reactions on protein scaffolds to explain these phenomena, wherein the enzyme-substrate-scaffold complex undergoes stochastic state switching to reach an active state. This model predicted anchored enzymatic reactions to be accelerated, amplified, and insulated from inhibition compared with those occurring in solution. We exploited a direct interaction between protein kinase C (PKC) and AKAP7α as a model to validate these predictions experimentally. Using a genetically encoded PKC activity reporter, we found that both the strength and speed of substrate phosphorylation were enhanced by AKAP7α. PKC tethered to AKAP7α was less susceptible to inhibition from the ATP-competitive inhibitor Gö6976 and the substrate-competitive inhibitor PKC 20-28, but not the activation-competitive inhibitor calphostin C. Model predictions and experimental validation demonstrated that insulation is a general property of scaffold tethering. Sensitivity analysis indicated that these findings may be applicable to many other scaffolds as well. Collectively, our findings provide theoretical and experimental evidence that scaffold proteins can amplify, accelerate, and insulate signal transduction.     

Protein kinase C-beta: An emerging connection between nutrient excess and obesity

There is currently a global epidemic of obesity as a result of recent changes in lifestyle. Excess body fat deposition is caused by an imbalance between energy intake and energy expenditure due to interactions between genetic and environmental factors. The signals and biological mechanisms that trigger fat accumulation by disrupting energy homeostasis are not well understood. There is considerable evidence now supporting a possible role of protein kinase C beta (PKCβ) in energy homeostasis. This review highlights recent findings on the role of PKCβ activation in the genesis and progression of obesity, and of PKCβ repression in mediating the beneficial effects of physical exercise. Available data support a model in which adipose PKCβ activation is among the initiating events that disrupt mitochondrial function through interaction with p66^shc and amplify fat accumulation and adipose dysfunction, with systemic consequences. Manipulation of PKCβ levels, activity, or signaling could provide a therapeutic approach to combat obesity and associated metabolic disorders.     

Protein Kinase D Is Implicated in the Reversible Commitment to Differentiation in Primary Cultures of Mouse Keratinocytes

Although commitment to epidermal differentiation is generally considered to be irreversible, differentiated keratinocytes (KCs) have been shown to maintain a regenerative potential and to reform skin epithelia when placed in a suitable environment. To obtain insights into the mechanism of reinitiation of this proliferative response in differentiated KCs, we examined the reversibility of commitment to Ca²⁺-induced differentiation. Lowering Ca²⁺ concentration to micromolar levels triggered culture-wide morphological and biochemical changes, as indicated by derepression of cyclin D1, reinitiation of DNA synthesis, and acquisition of basal cell-like characteristics. These responses were inhibited by Goedecke 6976, an inhibitor of protein kinase D (PKD) and PKCα, but not with GF109203X, a general inhibitor of PKCs, suggesting PKD activation by a PKC-independent mechanism. PKD activation followed complex kinetics with a biphasic early transient phosphorylation within the first 6 h, followed by a sustained and progressive phosphorylation beginning at 24 h. The second phase of PKD activation was followed by prolonged ERK1/2 signaling and progression to DNA synthesis in response to the low Ca²⁺ switch. Specific knockdown of PKD-1 by RNA interference or expression of a dominant negative form of PKD-1 did not have a significant effect on normal KC proliferation and differentiation but did inhibit Ca²⁺-mediated reinitiation of proliferation and reversion in differentiated cultures. The present study identifies PKD as a major regulator of a proliferative response in differentiated KCs, probably through sustained activation of the ERK-MAPK pathway, and provides new insights into the process of epidermal regeneration and wound healing.     

Mechanisms of Transient Signaling via Short and Long Prolactin Receptor Isoforms in Female and Male Sensory Neurons

Prolactin (PRL) regulates activity of nociceptors and causes hyperalgesia in pain conditions. PRL enhances nociceptive responses by rapidly modulating channels in nociceptors. The molecular mechanisms underlying PRL-induced transient signaling in neurons are not well understood. Here we use a variety of cell biology and pharmacological approaches to show that PRL transiently enhanced capsaicin-evoked responses involve protein kinase C ϵ (PKCϵ) or phosphatidylinositol 3-kinase (PI3K) pathways in female rat trigeminal (TG) neurons. We next reconstituted PRL-induced signaling in a heterologous expression system and TG neurons from PRL receptor (PRLR)-null mutant mice by expressing rat PRLR-long isoform (PRLR-L), PRLR-short isoform (PRLR-S), or a mix of both. Results show that PRLR-S, but not PRLR-L, is capable of mediating PRL-induced transient enhancement of capsaicin responses in both male and female TG neurons. However, co-expression of PRLR-L with PRLR-S (1:1 ratio) leads to the inhibition of the transient PRL actions. Co-expression of PRLR-L deletion mutants with PRLR-S indicated that the cytoplasmic site adjacent to the trans-membrane domain of PRLR-L was responsible for inhibitory effects of PRLR-L. Furthermore, in situ hybridization and immunohistochemistry data indicate that in normal conditions, PRLR-L is expressed mainly in glia with little expression in rat sensory neurons (3–5%) and human nerves. The predominant PRLR form in TG neurons/nerves from rats and humans is PRLR-S. Altogether, PRL-induced transient signaling in sensory neurons is governed by PI3K or PKCϵ, mediated via the PRLR-S isoform, and transient effects mediated by PRLR-S are inhibited by presence of PRLR-L in these cells.     

Xgravin-like (Xgl), a novel putative a-kinase anchoring protein (AKAP) expressed during embryonic development in Xenopus

A-kinase anchoring proteins (AKAPs) are a heterogeneous family of scaffolding proteins that regulate the compartmentalization of signaling components, in particular that of the broad specificity kinase PKA. Here we describe the identification of a new member of this gene family, termed Xenopus gravin-like (Xgl), which encodes a highly acidic protein of 268 kDa that shares extensive homology with human Gravin and murine SSeCKS. Xgl is zygotically expressed in a highly dynamic fashion. During gastrulation Xgl is expressed in posterior mesoderm of the dorsal blastopore lip. During neurulation expression is transiently detected in the forebrain, two bilateral neuroectodermal stripes and the notochord. At tailbud stages expression commences in the mandibular neural crest and the roof of the spinal cord from where neural crest cells migrate into the intersomitic region. In addition expression is detected in the heart and the anterior aspect of the chordoneural hinge.     

B-50/GAP-43 Phosphorylation and PKC Activity are Increased in Rat Hippocampal Synaptosomal Membranes After an Inhibitory Avoidance Training

Several lines of evidence indicate that protein kinase C (PKC) is involved in long-term potentiation (LTP) and in certain forms of learning. Recently, we found a learning-specific, time-dependent increase in [³H]phorbol dibutyrate binding to membrane-associated PKC in the hippocampus of rats subjected to an inhibitory avoidance task. Here we confirm and extend this observation, describing that a one trial inhibitory avoidance learning was associated with rapid and specific increases in B-50/GAP-43 phosphorylation in vitro and in PKC activity in hippocampal synaptosomal membranes. The increased phosphorylation of B-50/GAP-43 was seen at 30 min (+35% relative to naive or shocked control groups), but not at 10 or 60 min after training. This learning-associated increase in the phosphorylation of B-50/GAP-43 is mainly due to an increase in the activity of PKC. This is based on three different sets of data: 1) PKC activity increased by 24% in hippocampal synaptosomal membranes of rats sacrificed 30 min after training; 2) B-50/GAP-43 immunoblots revealed no changes in the amount of this protein among the different experimental groups; 3) phosphorylation assays, performed in the presence of bovine purified PKC or in the presence of the selective PKC inhibitor CGP 41231, exhibited no differences in B-50/GAP-43 phosphorylation between naive and trained animals. In conclusion, these results support the contention that hippocampal PKC participates in the early neural events of memory formation of an aversively-motivated learning task.