Stephen C. Parnell

A Systems-Biology Analysis of Feedback Inhibition in the Sho1 Osmotic-Stress-Response Pathway

BACKGROUND A common property of signal transduction systems is that they rapidly lose their ability to respond to a given stimulus. For instance in yeast, the mitogen-activated protein (MAP) kinase Hog1 is activated and inactivated within minutes, even when the osmotic-stress stimulus is sustained. RESULTS Here, we used a combination of experimental and computational analyses to investigate the dynamic behavior of Hog1 activation in vivo. Computational modeling suggested that a negative-feedback loop operates early in the pathway and leads to rapid attenuation of Hog1 signaling. Experimental analysis revealed that the membrane-bound osmosensor Sho1 is phosphorylated by Hog1 and that phosphorylation occurs on Ser-166. Moreover, Sho1 exists in a homo-oligomeric complex, and phosphorylation by Hog1 promotes a transition from the oligomeric to monomeric state. A phosphorylation-site mutation (Sho1(S166E)) diminishes the formation of Sho1-oligomers, dampens activation of the Hog1 kinase, and impairs growth in high-salt or sorbitol conditions. CONCLUSIONS These findings reveal a novel phosphorylation-dependent feedback loop leading to diminished cellular responses to an osmotic-stress stimulus.

The RACK1 ortholog Asc1 functions as a g-protein β subunit coupled to glucose responsiveness in yeast

According to the prevailing paradigm, G-proteins are composed of three subunits, an α subunit with GTPase activity and a tightly associated βγ subunit complex. In the yeast Saccharomyces cerevisiae there are two known Gα proteins (Gpa1 and Gpa2) but only one Gβγ, which binds only to Gpa1. Here we show that the yeast ortholog of RACK1 (receptor for activated protein kinase C1) Asc1 functions as the Gβ for Gpa2. As with other known Gβ proteins, Asc1 has a 7-WD domain structure, interacts directly with the Gα in a guanine nucleotide-dependent manner, and inhibits Gα guanine nucleotide exchange activity. In addition, Asc1 binds to the effector enzyme adenylyl cyclase (Cyr1), and diminishes the production of cAMP in response to glucose stimulation. Thus, whereas Gpa2 promotes glucose signaling through elevated production of cAMP, Asc1 has opposing effects on these same processes. Our findings reveal the existence of an unusual Gβ subunit, one having multiple functions within the cell in addition to serving as a signal transducer for cell surface receptors and intracellular effectors.

Genome-Scale Analysis Reveals Sst2 as the Principal Regulator of Mating Pheromone Signaling in the Yeast Saccharomyces cerevisiae

ABSTRACT A common property of G protein-coupled receptors is that they become less responsive with prolonged stimulation. Regulators of G protein signaling (RGS proteins) are well known to accelerate G protein GTPase activity and do so by stabilizing the transition state conformation of the G protein α subunit. In the yeast Saccharomyces cerevisiae there are four RGS-homologous proteins (Sst2, Rgs2, Rax1, and Mdm1) and two Gα proteins (Gpa1 and Gpa2). We show that Sst2 is the only RGS protein that binds selectively to the transition state conformation of Gpa1. The other RGS proteins also bind Gpa1 and modulate pheromone signaling, but to a lesser extent and in a manner clearly distinct from Sst2. To identify other candidate pathway regulators, we compared pheromone responses in 4,349 gene deletion mutants representing nearly all nonessential genes in yeast. A number of mutants produced an increase (sst2, bar1, asc1, and ygl024w) or decrease (cla4) in pheromone sensitivity or resulted in pheromone-independent signaling (sst2, pbs2, gas1, and ygl024w). These findings suggest that Sst2 is the principal regulator of Gpa1-mediated signaling in vivo but that other proteins also contribute in distinct ways to pathway regulation.

Periostin promotes renal cyst growth and interstitial fibrosis in polycystic kidney disease

In renal cystic diseases, sustained enlargement of fluid-filled cysts is associated with severe interstitial fibrosis and progressive loss of functioning nephrons. Periostin, a matricellular protein, is highly overexpressed in cyst-lining epithelial cells of autosomal dominant polycystic disease kidneys (ADPKD) compared to normal tubule cells. Periostin accumulates in situ within the matrix subjacent to ADPKD cysts, binds to αVβ3- and αVβ5-integrins and stimulates the integrin-linked kinase to promote cell proliferation. We knocked out periostin (Postn) in pcy/pcy mice, an orthologous model of nephronophthisis type 3, to determine whether periostin loss reduces PKD progression in a slowly progressive model of renal cystic disease. At 20 weeks of age, pcy/pcy: Postn−/− mice had a 34% reduction in kidney weight/body weight, a reduction in cyst number and total cystic area, a 69% reduction in phosphorylated S6, a downstream component of the mTOR pathway, and fewer proliferating cells in the kidneys compared to pcy/pcy: Postn+/+ mice. The pcy/pcy Postn knockout mice also had less interstitial fibrosis with improved renal function at 20 weeks and significantly longer survival (51.4 compared to 38.0 weeks). Thus, periostin adversely modifies the progression of renal cystic disease by promoting cyst epithelial cell proliferation, cyst enlargement and interstitial fibrosis, all contributing to the decline in renal function and premature death.

Combined computational and experimental analysis reveals mitogen-activated protein kinase-mediated feedback phosphorylation as a mechanism for signaling specificity.

A series of mathematical models was used to quantitatively characterize pheromone-stimulated kinase activation and determine how mitogen-activated protein (MAP) kinase specificity is achieved. The findings reveal how feedback phosphorylation of a common pathway component can limit the activity of a competing MAP kinase through feedback phosphorylation of a common activator, and thereby promote signal fidelity.

Combined computational and experimental analysis reveals MAP kinase-mediated feedback phosphorylation as a mechanism for signaling specificity

A series of mathematical models was used to quantitatively characterize pheromone-stimulated kinase activation and determine how mitogen-activated protein (MAP) kinase specificity is achieved. The findings reveal how feedback phosphorylation of a common pathway component can limit the activity of a competing MAP kinase through feedback phosphorylation of a common activator, and thereby promote signal fidelity.

Protein phosphatase-1α interacts with and dephosphorylates polycystin-1.

Polycystin signaling is likely to be regulated by phosphorylation. While a number of potential protein kinases and their target phosphorylation sites on polycystin-1 have been identified, the corresponding phosphatases have not been extensively studied. We have now determined that polycystin-1 is a regulatory subunit for protein phosphatase-1α (PP1α). Sequence analysis has revealed the presence of a highly conserved PP1-interaction motif in the cytosolic, C-terminal tail of polycystin-1; and we have shown that transfected PP1α specifically co-immunoprecipitates with a polycystin-1 C-tail construct. To determine whether PP1α dephosphorylates polycystin-1, a PKA-phosphorylated GST-polycystin-1 fusion protein was shown to be dephosphorylated by PP1α but not by PP2B (calcineurin). Mutations within the PP1-binding motif of polycystin-1, including an autosomal dominant polycystic kidney disease (ADPKD)-associated mutation, significantly reduced PP1α-mediated dephosphorylation of polycystin-1. The results suggest that polycystin-1 forms a holoenzyme complex with PP1α via a conserved PP1-binding motif within the polycystin-1 C-tail, and that PKA-phosphorylated polycystin-1 serves as a substrate for the holoenzyme.

Integrin-Linked Kinase Signaling Promotes Cyst Growth and Fibrosis in Polycystic Kidney Disease

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by innumerous fluid-filled cysts and progressive deterioration of renal function. Previously, we showed that periostin, a matricellular protein involved in tissue repair, is markedly overexpressed by cyst epithelial cells. Periostin promotes cell proliferation, cyst growth, interstitial fibrosis, and the decline in renal function in PKD mice. Here, we investigated the regulation of these processes by the integrin-linked kinase (ILK), a scaffold protein that links the extracellular matrix to the actin cytoskeleton and is stimulated by periostin. Pharmacologic inhibition or shRNA knockdown of ILK prevented periostin-induced Akt/mammalian target of rapamycin (mTOR) signaling and ADPKD cell proliferation in vitro Homozygous deletion of ILK in renal collecting ducts (CD) of Ilkfl/fl ;Pkhd1-Cre mice caused tubule dilations, apoptosis, fibrosis, and organ failure by 10 weeks of age. By contrast, Ilkfl/+ ;Pkhd1-Cre mice had normal renal morphology and function and survived >1 year. Reduced expression of ILK in Pkd1fl/fl ;Pkhd1-Cre mice, a rapidly progressive model of ADPKD, decreased renal Akt/mTOR activity, cell proliferation, cyst growth, and interstitial fibrosis, and significantly improved renal function and animal survival. Additionally, CD-specific knockdown of ILK strikingly reduced renal cystic disease and fibrosis and extended the life of pcy/pcy mice, a slowly progressive PKD model. We conclude that ILK is critical for maintaining the CD epithelium and renal function and is a key intermediate for periostin activation of signaling pathways involved in cyst growth and fibrosis in PKD.

A mutation affecting polycystin-1 mediated heterotrimeric G-protein signaling causes PKD

Abstract Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the growth of renal cysts that ultimately destroy kidney function. Mutations in the PKD1 and PKD2 genes cause ADPKD. Their protein products, polycystin-1 (PC1) and polycystin-2 (PC2) have been proposed to form a calcium-permeable receptor-channel complex; however the mechanisms by which they function are almost completely unknown. Most mutations in PKD1 are truncating loss-of-function mutations or affect protein biogenesis, trafficking or stability and reveal very little about the intrinsic biochemical properties or cellular functions of PC1. An ADPKD patient mutation (L4132Δ or ΔL), resulting in a single amino acid deletion in a putative G-protein binding region of the PC1 C-terminal cytosolic tail, was found to significantly decrease PC1-stimulated, G-protein-dependent signaling in transient transfection assays. Pkd1ΔL/ΔL mice were embryo-lethal suggesting that ΔL is a functionally null mutation. Kidney-specific Pkd1ΔL/cond mice were born but developed severe, postnatal cystic disease. PC1ΔL protein expression levels and maturation were comparable to those of wild type PC1, and PC1ΔL protein showed cell surface localization. Expression of PC1ΔL and PC2 complexes in transfected CHO cells failed to support PC2 channel activity, suggesting that the role of PC1 is to activate G-protein signaling to regulate the PC1/PC2 calcium channel.

Periostin overexpression in collecting ducts accelerates renal cyst growth and fibrosis in polycystic kidney disease

In polycystic kidney disease (PKD), persistent activation of cell proliferation and matrix production contributes to cyst growth and fibrosis, leading to progressive deterioration of renal function. Previously, we showed that periostin, a matricellular protein involved in tissue repair, is overexpressed by cystic epithelial cells of PKD kidneys. Periostin binds αVβ3-integrins and activates integrin-linked kinase (ILK), leading to Akt/mammalian target of rapamycin (mTOR)-mediated proliferation of human PKD cells. By contrast, periostin does not stimulate the proliferation of normal human kidney cells. This difference in the response to periostin is due to elevated expression of αVβ3-integrins by cystic cells. To determine whether periostin accelerates cyst growth and fibrosis, we generated mice with conditional overexpression of periostin in the collecting ducts (CDs). Ectopic CD expression of periostin was not sufficient to induce cyst formation or fibrosis in wild-type mice. However, periostin overexpression in pcy/pcy ( pcy) kidneys significantly increased mTOR activity, cell proliferation, cyst growth, and interstitial fibrosis; and accelerated the decline in renal function. Moreover, CD-specific overexpression of periostin caused a decrease in the survival of pcy mice. These pathological changes were accompanied by increased renal expression of vimentin, α-smooth muscle actin, and type I collagen. We also found that periostin increased gene expression of pathways involved in repair, including integrin and growth factor signaling and ECM production, and it stimulated focal adhesion kinase, Rho GTPase, cytoskeletal reorganization, and migration of PKD cells. These results suggest that periostin stimulates signaling pathways involved in an abnormal tissue repair process that contributes to cyst growth and fibrosis in PKD.