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Cascade of Events: A transmembrane receptor WITHOUT ENZYME ACTIVITY binds an extracellular chemical signal, causing a conformational change in the receptor which propagates through the membrane. The cycle of degradation and resynthesis of PIP2 is called the PI cycle.
Figure: PI cycle
Some signals that activate phospholipase C and make IP3 and diacylglycerol include: acetylcholine (a different class than the type located at the neuromuscular junction that we discussed in the last chapter section), angiotensin II, glutamate, histamine, oxytocin, platelet-derived growth factor, vasopressin, gonadotropin-releasing hormone, and thyrotropin-releasing hormone. Some proteins phosphorylated by PKC include:
Some kinases regulated by calcium and calmodulin include: myosin light chain kinase, PI-3 kinase, CAM-dependent kinases. Ca/CAM also regulates other proteins which include: adenylate cyclase (brain), Ca-dependent Na channel, cAMP phosphodiesterase, calcineurin (phosphoprotein phosphatase 2B), cAMP gated olfactory channels, NO synthase, and plasma membrane Ca/ATPase.
- Prof. Henry Jakubowski (College of St. Benedict/St. John's University)
Competitive regulation of CaT-like-mediated Ca 2+ entry by protein kinase C and calmodulin
A finely tuned Ca 2+ signaling system is essential for cells to transduce extracellular stimuli, to regulate growth, and to differentiate. We have recently cloned CaT-like (CaT-L), a highly selective Ca 2+ channel closely related to the epithelial calcium channels (ECaC) and the calcium transport protein CaT1. CaT-L is expressed in selected exocrine tissues, and its expression also strikingly correlates with the malignancy of prostate cancer. The expression pattern and selective Ca 2+ permeation properties suggest an important function in Ca 2+ uptake and a role in tumor progression, but not much is known about the regulation of this subfamily of ion channels. We now demonstrate a biochemical and functional mechanism by which cells can control CaT-L activity. CaT-L is regulated by means of a unique calmodulin binding site, which, at the same time, is a target for protein kinase C-dependent phosphorylation. We show that Ca 2+ -dependent calmodulin binding to CaT-L, which facilitates channel inactivation, can be counteracted by protein kinase C-mediated phosphorylation of the calmodulin binding site.
Tight regulation of intracellular Ca 2+ homeostasis is essential for the survival of virtually all cell types. Although much is known about the components and their physiological function maintaining rapid Ca 2+ signaling in excitable tissue (1, 2), less is known in nonexcitable cells. Although there is growing evidence for changes in Ca 2+ -homeostasis causing cells to proliferate and ultimately become malignant cancer cells (3), the molecular components that cause these changes are largely unknown. A subfamily of Ca 2+ -selective ion channels has recently emerged that is involved in transcellular Ca 2+ uptake in epithelial cells (4, 5), and its members are very distantly related to transient receptor potential (TRP)-channels. In contrast to the activation mechanism discussed for some of the classic members of the mammalian TRP family (6, 7), these epithelial channels are not likely to be gated by depletion of internal calcium stores (5, 8). We have recently identified a member of this family, human calcium transport protein-like (CaT-L), and demonstrated that its physiological profile concerning current size and Ca 2+ selectivity resembles that of rabbit epithelial calcium channels (ECaC) (9). Its sequence is almost identical to the also recently cloned human CaT1 (10), but its expression pattern in healthy trophoblasts and syncytiotrophoblasts of the placenta, pancreatic acinar cells, and salivary glands, but not in kidney or small intestine differs from rabbit ECaC and rat CaT1 beyond the expected species differences. Interestingly, we found that, whereas CaT-L expression cannot be detected in normal prostate tissues, its expression increases when these tissues undergo malignant transformation to metastatic stages that infiltrate the rest of the body (9).
The expression pattern and the selective Ca 2+ permeation properties of CaT-L channels suggest an important function in Ca 2 + uptake and possibly in the potential for cellular transformation. The regulation and modulation of the related ECaC are not well characterized, and, given the fact that CaT-L shows very low overall homology (13–19% sequence identity) to the better characterized members of the “classic” TRP family, such as Drosophila TRP and TRPL (for review see refs. 11–13), we investigated feedback regulation of CaT-L channel activity. We show that inactivation of CaT-L is a multiphasic process with a rapid calcium-dependent phase and a later calcium-calmodulin (Ca 2+ -CaM)-dependent phase. We further demonstrate by biochemical and functional analyses that the calmodulin-dependent inactivation can be counteracted by phosphorylation through protein kinase C (PKC).
Protocadherins (Pcdhs) belong to the cadherin superfamily, a group of cell adhesion molecules that are known to play critical roles in several biological processes, including embryonic morphogenesis, neural circuit formation, angiogenesis, and cancer 1,2,3 . The clustered Pcdhs, consisting of
60 proteins encoded by three tandem gene clusters (Pcdha, Pcdhb, and Pcdhg) on human chromosome 5q31 (chromosome 18 in the mouse), represent the largest subgroup within the cadherin superfamily 4,5 . Within the Pcdha and Pcdhg clusters, large “variable” exons encoding 6 extracellular cadherin (EC) repeats, a transmembrane domain, and a variable cytoplasmic domain (VCD) are expressed from their own promoters and spliced to three short constant exons that encode a C-terminal domain shared by all α- or γ-Pcdhs (β-Pcdhs do not have this shared domain and are thus each encoded by a single exon Pcdhg cluster schematized in Fig. 1a) 4,6,7 . Clustered Pcdhs, as a group, are strongly expressed throughout the developing and mature nervous system 8,9,10,11,12 , with lower levels detected in other organs such as lung and kidney 13,14 . The expression of individual clustered Pcdh genes is differentially regulated in each cell via promoter methylation and interaction with distant regulatory elements 15,16,17,18 . Single-cell RT-PCR analysis suggests that each neuron expresses a fraction of the 14 Pcdha, 22 Pcdhb, and 22 Pcdhg genes most are stochastically and sparsely expressed, though 5 “C” subtype genes (Pcdhac1, Pcdhac2, Pcdhgc3, Pcdhgc4, and Pcdhgc5) appear to be expressed ubiquitously 19,20,21 . All clustered Pcdhs form cis-multimers that interact in a strictly homophilic manner in trans 22,23,24 .
(a) Schematic of the mouse Pcdhg gene cluster (top), with examples of promoter activation, gene transcription, and splicing. Example transcripts and resultant proteins are schematized below. Each of the 22 isoforms is encoded by 4 exons: 1 variable exon (shades of purple or blue encodes 6 EC domains, a transmembrane domain, and a VCD) and 3 constant exons (black encode a shared C-terminal domain). EC: extracellular cadherin repeats VCD: variable cytoplasmic domain CON: constant region. (b) TOPFlash assays were performed by co-transfecting plasmids encoding individual γ-Pcdh isoforms with the SUPER-TOPFlash and Renilla luciferase plasmids into HEK293 cells. Cell lysates were assayed for luciferase activity 24 hours after exposure to Wnt3a CM. Results are presented as a “Wnt Response Index”, defined as the fold change in Relative Luciferase Units (RLU ratio of firefly luciferase to Renilla luciferase [control for transfection efficiency]) following Wnt treatment in cells transfected with individual γ-Pcdh isoforms compared to that in cells expressing only a GFP negative control. Thus, the control (GFP only) Wnt cellular response is the baseline (“0”), and manipulations that increase further that response yield a positive index, while those that decrease that response yield a negative index. Several γ-Pcdh isoforms significantly upregulated Wnt signalling activity, while only γ-Pcdh-C3 significantly downregulated Wnt signalling. Data are presented on a log2 scale of means ± SEM from 6 independent experiments. A two-way ANOVA with Bonferroni post hoc test (to correct for multiple comparisons) was performed to assess statistical significance. *p < 0.05, and **p < 0.01.
The clustered Pcdhs play critical roles in neurodevelopment. Using constitutive and conditional Pcdhg mouse mutants, we demonstrated that the γ-Pcdhs regulate neuronal survival 11,25 , synaptogenesis 26,27 , astrocyte-neuron interactions 26 , dendrite arborisation 8,28,29 , and axonal patterning 30 . In retinal starburst amacrine cells, the γ-Pcdhs are also required for normal dendrite self-avoidance 31,32 , underscoring the importance of this family for the proper formation of dendrite arbours. The relative importance of the γ-Pcdhs is underscored by the fact that mice lacking all 22 isoforms die shortly after birth 7 , in contrast to the viable and fertile Pcdha cluster nulls 33 . Nevertheless, the α-Pcdhs have been shown to regulate dendrite arborisation and dendritic spine formation 34 , as well as axonal targeting 33,35,36,37 , in a variety of neurons.
Intriguingly, the γ-Pcdhs also have been recently implicated as potential tumour suppressors, via inhibition of canonical Wnt signalling 13 . A genome-wide analysis of promoter hypermethylation in Wilms’ tumour, a paediatric kidney cancer, identified the clustered Pcdh genes as consistently hypermethylated many of them, especially those encoding γ-Pcdhs, are indeed transcriptionally silenced in tumour cells. Knockdown of all Pcdhg genes (using an siRNA targeting the constant region) in kidney cell lines led to increased canonical Wnt signalling, while overexpression of individual Pcdhg cDNAs inhibited the Wnt pathway and led to reduced tumour cell growth in soft agar assays 13 . The ubiquitously-expressed γ-Pcdh-C3 isoform (encoded by Pcdhgc3) was similarly shown to be silenced in colorectal cancer cells, and to reduce growth of these cells when overexpressed via inhibition of a Wnt-mTOR pathway 38 . Together with other studies identifying aberrant methylation of Pcdhga11 in astrocytomas 39 , Pcdhb genes in neuroblastoma 40 , and Pcdhg genes in toxicant-induced malignant cells 41 , these data suggest that understanding how clustered Pcdhs regulate tumour growth pathways will be of future translational importance. Intriguingly, a non-clustered δ-Pcdh known as PAPC/Pcdh8/Arcadlin can also affect canonical 42 and non-canonical 43,44 Wnt pathways, suggesting that Pcdhs in general may be of interest to our understanding of Wnt biology.
Here, we have investigated the molecular mechanisms through which γ-Pcdh-C3 inhibits canonical Wnt signalling, a well-known tumourigenic pathway that is also of paramount importance for many steps in neurodevelopment 45,46 . Binding of Wnt ligands to Frizzled and the Lrp5/6, Ryk, or ROR co-receptors can activate at least three distinct pathways: the canonical (β-catenin/TCF), PCP (planar cell polarity), and Wnt-Ca2 + pathways 47,48,49 . In the canonical pathway, β-catenin is found in a complex including Axin1, Adenomatous Polyposis Coli (APC) and GSK3β in the absence of Wnt ligand (the OFF state reviewed in Clevers and Nusse, 2012) 50 . Axin1 acts as a scaffold for the “destruction complex”: GSK3β phosphorylates β-catenin, which is then targeted for ubiquitin-dependent degradation by the proteasome. In the presence of the Wnt ligand (the ON state), Dishevelled (Dvl) binds at the C-terminus of Frizzled 51 and recruits Axin1 to the cytoplasmic tail of Lrp5/6, facilitating the phosphorylation of Lrp5/6 by GSK3β and CK1 52,53,54,55,56,57 . Phospho-Lrp6 is thought to directly inhibit GSK3β activity in the destruction complex, leading to reduced phosphorylation of β-catenin as well as Axin1 itself. Dephosphorylated Axin1 dissociates from the activated receptor complex and also from β-catenin, thus inactivating the destruction complex until Axin1 is again phosphorylated 55,58 . This inactivation of the destruction complex is thought to allow β-catenin to accumulate in the cytoplasm and to translocate to the nucleus. There, it displaces Groucho/TLE corepressors bound to TCF (T-cell factor)/LEF (Lymphoid enhancer factor) transcription factors, thereby activating Wnt target genes, including many that promote tumour proliferation or regulate brain patterning, dendrite arborisation, and synaptogenesis 45,50,59 .
Though the γ-Pcdhs have been implicated in the regulation of Wnt signalling 13,38 , the underlying molecular mechanisms are unknown. Here, we show that γ-Pcdh-C3, but not other γ-Pcdhs, significantly inhibits exogenous activation of canonical Wnt signalling in cultured cells, and identify the C3-specific VCD as the critical site of action. Though it has been reported that the intracellular domain of γ-Pcdhs can be cleaved and trafficked to the nucleus 60,61 , we show that the C3 VCD acts at the membrane, not the nucleus, to inhibit Wnt signalling. We further show that the C3 VCD physically, and directly, interacts with the DIX domain of Axin1, and present evidence that it competes for this binding site with Dvl1. Binding of Axin1 to the C3 VCD stabilizes Axin1 at the membrane and prevents Lrp6 phosphorylation, suggesting that in this context, membrane localization can actually stabilize the β-catenin destruction complex. Finally, we use a Wnt signalling reporter mouse line and an inducible γ-Pcdh-C3 overexpression allele to show that that increasing C3 levels reduces endogenous Wnt signalling in the mouse cerebral cortex in vivo. Together, these data identify a novel molecular mechanism by which γ-Pcdhs can affect Wnt signalling, and identify Axin1 as the first known intracellular signalling partner for the widely-expressed γ-Pcdh-C3 isoform.
Caspase-3-mediated cleavage of ROCK I induces MLC phosphorylation and apoptotic membrane blebbing
Increased phosphorylation of myosin light chain (MLC) is necessary for the dynamic membrane blebbing that is observed at the onset of apoptosis. Here we identify ROCK I, an effector of the small GTPase Rho, as a new substrate for caspases. ROCK I is cleaved by caspase-3 at a conserved DETD1113/G sequence and its carboxy-terminal inhibitory domain is removed, resulting in deregulated and constitutive kinase activity. ROCK proteins are known to regulate MLC-phosphorylation, and apoptotic cells exhibit a gradual increase in levels of phosphorylated MLC concomitant with ROCK I cleavage. This phosphorylation, as well as membrane blebbing, is abrogated by inhibition of caspases or ROCK proteins, but both processes are independent of Rho activity. We also show that expression of active truncated ROCK I induces cell blebbing. Thus, activation of ROCK I by caspase-3 seems to be responsible for bleb formation in apoptotic cells.
Neuroglia, the “glue” that fills the space between neurons in the central nervous system, takes active part in nerve cell signaling. Neuroglial cells, astroglia, oligodendroglia, and microglia, are together about as numerous as neurons in the brain as a whole, and in the cerebral cortex grey matter, but the proportion varies widely among brain regions. Glial volume, however, is less than one-fifth of the tissue volume in grey matter. When stimulated by neurons or other cells, neuroglial cells release gliotransmitters by exocytosis, similar to neurotransmitter release from nerve endings, or by carrier-mediated transport or channel flux through the plasma membrane. Gliotransmitters include the common neurotransmitters glutamate and GABA, the nonstandard amino acid d -serine, the high-energy phosphate ATP, and l -lactate. The latter molecule is a “buffer” between glycolytic and oxidative metabolism as well as a signaling substance recently shown to act on specific lactate receptors in the brain. Complementing neurotransmission at a synapse, neuroglial transmission often implies diffusion of the transmitter over a longer distance and concurs with the concept of volume transmission. Transmission from glia modulates synaptic neurotransmission based on energetic and other local conditions in a volume of tissue surrounding the individual synapse. Neuroglial transmission appears to contribute significantly to brain functions such as memory, as well as to prevalent neuropathologies.