Stimulation of phospholipase Cß1 by Gαq promotes the assembly of stress granule proteins.

During adverse conditions, mammalian cells suppress protein production by sequestering the translational machinery in membrane-less organelles known as stress granules. Here, we found that activation of the G protein subunit Gαq promoted the formation of particles that contained stress granule proteins through a mechanism linked to a cytosolic fraction of phospholipase Cß1 (PLCß1). In experiments with PC12 and A10 cells, we showed that under basal conditions, cytosolic PLCß1 bound to stress granule­associated proteins, including PABPC1, eIF5A, and Ago2. Knockdown of cytosolic PLCß1 with siRNA or promoting its relocalization to the plasma membrane by activating Gαq resulted in the formation of particles containing these stress granule­associated proteins. Our studies showed that the composition of these particles resembled those formed under osmotic stress and were distinct from those formed in response to other types of stress. Our results fit a simple thermodynamic model in which cytosolic PLCß1 solubilizes stress granule proteins such that its movement to activated Gαq releases these proteins to enable the formation of stress granules. Together, our data suggest a link between Gαq-coupled signals and protein translation through stress granule formation.

The chromatin-binding domain of Ki-67 together with p53 protects human chromosomes from mitotic damage.

Vertebrate mammals express a protein called Ki-67 which is most widely known as a clinically useful marker of highly proliferative cells. Previous studies of human cells indicated that acute depletion of Ki-67 can elicit a delay at the G1/S boundary of the cell cycle, dependent on induction of the checkpoint protein p21. Consistent with those observations, we show here that acute Ki-67 depletion causes hallmarks of DNA damage, and the damage occurs even in the absence of checkpoint signaling. This damage is not observed in cells traversing S phase but is instead robustly detected in mitotic cells. The C-terminal chromatin-binding domain of Ki-67 is necessary and sufficient to protect cells from this damage. We also observe synergistic effects when Ki-67 and p53 are simultaneously depleted, resulting in increased levels of chromosome bridges at anaphase, followed by the appearance of micronuclei. Therefore, these studies identify the C terminus of Ki-67 as an important module for genome stability.

Stimulation of the Gαq/phospholipase Cß1 signaling pathway returns differentiated cells to a stem-like state.

Phospholipase Cß1 is activated by Gαq to generate calcium signals in response to hormones and neurotransmitters. Besides carrying out this plasma membrane function, PLCß1 has a cytosolic population that helps to drive the differentiation of PC12 cells by inhibiting a nuclease that promotes RNA-induced silencing (C3PO). Here, we show that down-regulating PLCß1 or reducing its cytosolic population by activating Gαq to localize it to the plasma membrane returns differentiated PC12 and SK-N-SH cells to an undifferentiated state. In this state, PC12 cells have a spherical morphology, resume proliferation, and express the stem cell transcription factors nanog and Oct4. Similar changes are seen when C3PO is down-regulated. This return to a stem-like state is accompanied by shifts in multiple miR populations. Surprisingly, de-differentiation can be induced by extended stimulation of Gαq where cells return to a spherical morphology and levels of specific miRs return to their undifferentiated values. In complementary studies, we followed the real-time hydrolysis of a fluorescent-tagged miR in cells where PLCß1 or C3PO were down-regulated in PC12 cells and find substantial differences in miR processing in the undifferentiated and differentiated states. Taken together, our studies suggest that PLCß1, through its ability to regulate C3PO and endogenous miR populations, mediates the differentiation of two types of cultured neuronal cells.

The Gαq/phospholipase Cß signaling system represses tau aggregation.

Alzheimer's disease is typified by calcium dysfunction and neurofibrillary tangles of tau aggregates along with mitotic proteins. Using PC12 cells as a model system, we determined whether the Gαq/PLCß/ calcium signaling pathway impacts the manifestation of Alzheimer's disease. Down-regulating PLCß significantly increases tau protein expression and causes a large increase in tau aggregation. Stimulating Gαq to activate PLCß results in a modest reduction in tau aggregation while inhibiting PLCß activity results in a modest enhancement of tau aggregation. These results suggest that PLCß may effect tau aggregation by an additional mechanism that is independent of its ability to transduce calcium signals. To this end, we found that a cytosolic population of PLCß binds to a mitotic protein found in neurofibrillary tangles, CDK18, which promotes tau phosphorylation and aggregation. Taken together, our studies show that the loss of PLCß1 can promote Alzheimer's disease by a combination of its catalytic activity and its interaction with mitotic proteins thus offering an orthogonal method to control tau aggregation.

Mechanical Stretch Redefines Membrane Gαq-Calcium Signaling Complexes.

Muscle cells are routinely subjected to mechanical stretch but the impact of stretch on the organization of membrane domains is unknown. In this study, we characterize the effect of stretch on GPCR-Gαq protein signaling. Activation of this pathway leads to an increase in intracellular calcium. In muscle cells, GPCR-Gαq signals are enhanced when these proteins are localized in caveolae membrane domains whose curved structure can flatten with stretch. When we statically stretch rat aortic smooth muscle A10 cells by 1-5%, cellular calcium appears unperturbed as indicated by a calcium indicator. However, when we activate the bradykinin type 2 receptor (B2R)/Gαq pathway, we observe a loss in calcium that appears to be mediated through perturbations in calcium-activated stretch receptors. In contrast, if we apply oscillating stretch, calcium levels are enhanced. We tested whether the observed changes in B2R-Gαq calcium signals were caused by stretch-induced disruption of caveolae using a combination of silencing RNA technology and growth conditions. We find that stretch changes the ability of monoclonal caveolin antibodies to bind caveolae indicating a change in configuration of the domains. This change is seen by the inability of cells to survive stretch cycles when the level of caveolae is significantly reduced. Our studies show that the effect of calcium signals by mechanical stretch is mediated by the type of stretch and the amount of caveolae.

Phospholipase Cß1 regulates proliferation of neuronal cells.

Cells have developed lineage-specific mechanisms to control proliferation and drive morphologic changes upon differentiation. A hallmark of differentiation is the assembly of signaling molecules that transduce extracellular signals, such as the production of the G protein-regulated enzyme phospholipase Cß (PLCß), which generates calcium signals from sensory stimuli. We found that in most cancerous cell lines there is positive correlation between PLCß1 levels and cell proliferation. In cells of neuronal lineage, however, reducing PLCß1 levels increases the rate of proliferation. Using a combination of biochemical and biophysical methods, we find that, in the G1 phase, a cytosolic population of PLCß1 associates with cyclin-dependent kinase 16 (CDK16), a neuron-specific enzyme that is activated by cyclin Y to inactivate the antioncogenic protein p27Kip1. Binding of PLCß1 directly inhibits CDK16 activity and in turn reduces the ability of cells to enter the S phase. Activation of Gαq by carbachol causes movement of PLCß from the cytosol to the plasma membrane, reducing its association with CDK16. Similarly, the overexpression of activated Gαq moves PLCß1 to the membrane, reverses G1 arrest, and promotes proliferation, thereby connecting external stimuli with cell proliferation. Our results present a model in which the transient high expression of PLCß1 that occurs at the onset of differentiation arrests cells in the G1 phase through its association with CDK16 and allows CDK16 to transition to its postmitotic function of neurite outgrowth and trafficking of synaptic vesicles. The novel role of PLCß1 in neuronal cell proliferation offers a unique interaction that can be manipulated to guide cells into a neuronal phenotype or to develop therapies for neuroblastomas.-Garwain, O., Valla, K., Scarlata, S. Phospholipase Cß1 regulates proliferation of neuronal cells.

Phospholipase Cß interacts with cytosolic partners to regulate cell proliferation.

Phospholipase Cß (PLCß) is the main effector of the Gαq signaling pathway relaying different extracellular sensory information to generate intracellular calcium signals. Besides this classic function, we have found that PLCß plays an important but unknown role in regulating PC12 cell differentiation by interacting with components in the RNA-induced silencing machinery. In trying to understand the role of PLCß in PC12 cell differentiation, we find that over-expressing PLCß reduces PC12 cell proliferation while down-regulating PLCß increases the rate of cell proliferation. However, this behavior is not seen in other cancerous cell lines. To determine the underlying mechanism, we carried out mass spectrometry analysis of PLCß complexes in PC12 cells. We find that in unsynchronized cells, PLCß primarily binds cyclin-dependent kinase (CDK)16 whose activity plays a key role in cell proliferation. In vitro studies show a direct association between the two proteins that result in loss in CDK16 activity. When cells are arrested in the G2/M phase, a large population of PLCß is bound to Ago2 in a complex that contains C3PO and proteins commonly found in stress granules. Additionally, another population of PLCß complexes with CDK18 and cyclin B1. Fluorescence lifetime imaging microscopy (FLIM) confirms cell cycle dependent associations between PLCß and these other protein binding partners. Taken together, our studies suggest that PLCß may play an active role in mediating interactions required to move through the cell cycle.

Phospholipase Cß-TRAX Association Is Required for PC12 Cell Differentiation.

When treated with nerve growth factor, PC12 cells will differentiate over the course of several days. Here, we have followed changes during differentiation in the cellular levels of phosphoinositide-specific phospholipase Cß (PLCß) and its activator, Gαq, which together mediate Ca2+ release. We also followed changes in the level of the novel PLCß binding partner TRAX (translin-associated factor X), which promotes RNA-induced gene silencing. We find that the level of PLCß increases 4-fold within 24 h, whereas Gαq increases only 1.4-fold, and this increase occurs ∼24 h later than PLCß. Alternately, the level of TRAX remains constant over the 72 h tested. When PLCß1 or TRAX is down-regulated, differentiation does not occur. The impact of PLCß on differentiation appears independent of Gαq as down-regulating Gαq at constant PLCß does not affect differentiation. Förster resonance energy transfer studies after PLCß association with its partners indicate that PLCß induced soon after nerve growth factor treatment associates with TRAX rather than Gαq Functional measurements of Ca2+ signals to assess the activity of PLCß-Gαq complexes and measurements of the reversal of siRNA(GAPDH) to assess the activity of PLCß-TRAX complexes additionally suggest that the newly synthesized PLCß associates with TRAX to impact RNA-induced silencing. Taken together, our studies show that PLCß, through its ability to bind TRAX and reverse RNA silencing of specific genes, plays a key role in switching PC12 cells to their differentiated state.

Phospholipase Cß connects G protein signaling with RNA interference.

Phosphoinositide-specific-phospholipase Cß (PLCß) is the main effector of Gαq stimulation which is coupled to receptors that bind acetylcholine, bradykinin, dopamine, angiotensin II as well as other hormones and neurotransmitters. Using a yeast two-hybrid and other approaches, we have recently found that the same region of PLCß that binds Gαq also interacts with Component 3 Promoter of RNA induced silencing complex (C3PO), which is required for efficient activity of the RNA-induced silencing complex. In purified form, C3PO competes with Gαq for PLCß binding and at high concentrations can quench PLCß activation. Additionally, we have found that the binding of PLCß to C3PO inhibits its nuclease activity leading to reversal of RNA-induced silencing of specific genes. In cells, we found that PLCß distributes between the plasma membrane where it localizes with Gαq, and in the cytosol where it localizes with C3PO. When cells are actively processing small interfering RNAs the interaction between PLCß and C3PO gets stronger and leads to changes in the cellular distribution of PLCß. The magnitude of attenuation is specific for different silencing RNAs. Our studies imply a direct link between calcium responses mediated through Gαq and post-transcriptional gene regulation through PLCß.