Phosphorylation Of Key Residues Research Articles

Divergent Effects of G2019S and R1441C LRRK2 Mutations on LRRK2 and Rab10 Phosphorylations in Mouse Tissues

Mutations in LRRK2 cause familial Parkinson’s disease and common variants increase disease risk. LRRK2 kinase activity and cellular localization are tightly regulated by phosphorylation of key residues, primarily Ser1292 and Ser935, which impacts downstream phosphorylation of its substrates, among which Rab10. A comprehensive characterization of LRRK2 activity and phosphorylation in brain as a function of age and mutations is missing. Here, we monitored Ser935 and Ser1292 phosphorylation in midbrain, striatum, and cortex of 1, 6, and 12 months-old mice carrying G2019S and R1441C mutations or murine bacterial artificial chromosome (BAC)-Lrrk2-G2019S. We observed that G2019S and, at a greater extent, R1441C brains display decreased phospho-Ser935, while Ser1292 autophosphorylation increased in G2019S but not in R1441C brain, lung, and kidney compared to wild-type. Further, Rab10 phosphorylation, is elevated in R1441C carrying mice, indicating that the effect of LRRK2 mutations on substrate phosphorylation is not generalizable. In BAC-Lrrk2-G2019S striatum and midbrain, Rab10 phosphorylation, but not Ser1292 autophosphorylation, decreases at 12-months, pointing to autophosphorylation and substrate phosphorylation as uncoupled events. Taken together, our study provides novel evidence that LRRK2 phosphorylation in mouse brain is differentially impacted by mutations, brain area, and age, with important implications as diagnostic markers of disease progression and stratification.

New aspects in cardiac L-type Ca2+ channel regulation.

Cardiac excitation-contraction coupling is initiated with the influx of Ca2+ ions across the plasma membrane through voltage-gated L-type calcium channels. This process is tightly regulated by modulation of the channel open probability and channel localization. Protein kinase A (PKA) is found in close association with the channel and is one of the main regulators of its function. Whether this kinase is modulating the channel open probability by phosphorylation of key residues or via alternative mechanisms is unclear. This review summarizes recent findings regarding the PKA-mediated channel modulation and will highlight recently discovered regulatory mechanisms that are independent of PKA activity and involve protein-protein interactions and channel localization.

A Novel Phosphoregulatory Switch Controls the Activity and Function of the Major Catalytic Subunit of Protein Kinase A in Aspergillus fumigatus.

ABSTRACTInvasive aspergillosis (IA), caused by the filamentous fungal pathogen Aspergillus fumigatus, is a major cause of death among immunocompromised patients. The cyclic AMP/protein kinase A (PKA) signaling pathway is essential for hyphal growth and virulence of A. fumigatus, but the mechanism of regulation of PKA remains largely unknown. Here, we discovered a novel mechanism for the regulation of PKA activity in A. fumigatus via phosphorylation of key residues within the major catalytic subunit, PkaC1. Phosphopeptide enrichment and tandem mass spectrometry revealed the phosphorylation of PkaC1 at four sites (S175, T331, T333, and T337) with implications for important and diverse roles in the regulation of A. fumigatus PKA. While the phosphorylation at one of the residues (T333) is conserved in other species, the identification of three other residues represents previously unknown PKA phosphoregulation in A. fumigatus. Site-directed mutagenesis of the phosphorylated residues to mimic or prevent phosphorylation revealed dramatic effects on kinase activity, growth, conidiation, cell wall stress response, and virulence in both invertebrate and murine infection models. Three-dimensional structural modeling of A. fumigatus PkaC1 substantiated the positive or negative regulatory roles for specific residues. Suppression of PKA activity also led to downregulation of PkaC1 protein levels in an apparent novel negative-feedback mechanism. Taken together, we propose a model in which PkaC1 phosphorylation both positively and negatively modulates its activity. These findings pave the way for future discovery of fungus-specific aspects of this key signaling network.

Estrogen Receptor and Receptor Tyrosine Kinase Signaling: Use of Combinatorial Hormone and Epidermal Growth Factor Receptor/Human Epidermal Growth Factor Receptor 2–Targeted Therapies for Breast Cancer

Estrogen receptor (ER ) is expressed in nearly 70% of breast cancers and is a key driver of growth in ER-positive breast tumors. ER is primarily a nuclear protein that, when activated by estrogens, acts as a ligand-dependent transcription factor and promotes the expression of several genes that enhance cell survival, proliferation, and tumor progression (Fig 1). Examples of gene products upregulated by ER’s genomic actions include Bcl-2, cyclin D1, and the insulin-like growth factor 1 (IGF-1) receptor. Activated ER can also be localized to the cellular membrane and exert rapid, nongenomic effects. Here ER can colocalize with numerous different signaling molecules that lead to activation of growth and survival-promoting pathways, including the mitogen-activated protein kinase (MAPK) and Akt pathways. The use of hormone therapies, including aromatase inhibitors and selective estrogen receptor modulators, that target and downregulate ER signaling in the breast is a mainstay of treatment for patients with ER-positive breast cancers. Although these agents are extremely effective, a significant proportion of patients exhibit de novo or acquired resistance to hormone therapy. A proposed mechanism of resistance to hormone therapy is signaling crosstalk between ER and growth factor receptor tyrosine kinases (RTKs). In an accompanying article, Guarneri et al report the results of a randomized, double-blind, placebo-controlled, multicenter phase IIb neoadjuvant study of the aromatase inhibitor letrozole combined with the dual epidermal growth factor receptor/human epidermal growth factor receptor 2 (EGFR/ HER2) inhibitor lapatinib in postmenopausal hormone receptor– positive, HER2-negative early-stage breast cancer. Their results show that clinical response rates were similar for patients who received a combination of letrozole and lapatinib compared with patients receiving letrozole and placebo (70% v 63%, respectively). However, on further subset analysis, they found that patients with a PIK3CA mutation (37%) had a significantly higher likelihood of clinical response to the letrozole-lapatinib combination therapy versus those patients without PIK3CA mutations (overall response rate of 93% for patients with mutant PIK3CA v 63% for patients with wild-type PIK3CA). PIK3CA is the gene that encodes the PI3K catalytic subunit p110 , and the PI3K pathway is the most commonly mutated pathway in breast cancers, with PIK3CA recurrent hotspot mutations found at high frequency in ER-positive breast tumors. PI3K has been implicated as a key mediator of hormone resistance in preclinical models, and evidence suggests that hyperactivation of PI3K may enhance estrogen-independent and -dependent ER transcriptional activity. Although inhibition of the PI3K pathway has been postulated to sensitize cells to hormone therapy, several retrospective clinical studies have shown that activating mutations in PIK3CA paradoxically predict for a better long-term prognosis in patients with ER-positive breast cancers treated with hormonal therapy although other studies have shown contradictory results. It is known that activated RTKs and their downstream signaling partners can directly interact with ER and cause phosphorylation of key residues in the AF-1 domain of ER. This phosphorylation results in activation of ER in a ligand-independent fashion to promote cell growth and survival. In addition, membrane-activated ER can activate the EGFR pathway by increasing the production of the EGFR ligand heparin-binding epidermal growth factor. Furthermore, in vitro studies have shown that breast cancer cell lines with acquired resistance to hormone therapy have increased expression and activation of EGFR and HER2 along with their downstream signaling pathways. The use of ErbB-targeted tyrosine kinase inhibitors or the anti-HER2 monoclonal antibody trastuzumab significantly inhibits the growth of these cells after development of resistance to hormone therapies. In addition, sensitivity to hormone therapies can be restored when resistant cells are treated with lapatinib, a dual EGFR/ HER2 inhibitor. Together, these data suggest that enhanced RTK signaling and activationofPI3Ksignalingmaybekeycontributorstoresistancetohormone therapies in breast cancer. This provides the rationale for the dual JOURNAL OF CLINICAL ONCOLOGY U N D E R S T A N D I N G T H E P A T H W A Y VOLUME 32 NUMBER 10 APRIL 1 2014

Activation of group I metabotropic glutamate receptors potentiates heteromeric kainate receptors.

Kainate receptors (KARs), a family of ionotropic glutamate receptors, are widely expressed in the central nervous system and are critically involved in synaptic transmission. KAR activation is influenced by metabotropic glutamate receptor (mGlu) signaling, but the underlying mechanisms are not understood. We undertook studies to examine how mGlu modulation affects activation of KARs. Confocal immunohistochemistry of rat hippocampus and cultured rat cortex revealed colocalization of the high-affinity KAR subunits with group I mGlu receptors. In hippocampal and cortical cultures, the calcium signal caused by activation of native KARs was potentiated by activation of group I mGlu receptors. In Xenopus laevis oocytes, activation of group I mGlu receptors potentiated heteromeric but not homomeric KAR-mediated currents, with no change in agonist potency. The potentiation of heteromeric KARs by mGlu1 activation was attenuated by GDPβS, blocked by an inhibitor of phospholipase C or the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), prolonged by the phosphatase inhibitor okadaic acid, but unaffected by the tyrosine kinase inhibitor lavendustin A. Protein kinase C (PKC) inhibition reduced the potentiation by mGlu1 of GluK2/GluK5, and conversely, direct activation of PKC by phorbol 12-myristate,13-acetate potentiated GluK2/GluK5. Using site-directed mutagenesis, we identified three serines (Ser833, Ser836, and Ser840) within the membrane proximal region of the GluK5 C-terminal domain that, in combination, are required for mGlu1-mediated potentiation of KARs. Together, these data suggest that phosphorylation of key residues in the C-terminal domain changes the overall charge of this domain, resulting in potentiated agonist responses.

Protein Kinase C Phosphorylation Regulates Membrane Insertion of GABAA Receptor Subtypes That Mediate Tonic Inhibition

Tonic inhibition in the brain is mediated largely by specialized populations of extrasynaptic receptors, γ-aminobutyric acid receptors (GABA(A)Rs). In the dentate gyrus region of the hippocampus, tonic inhibition is mediated primarily by GABA(A)R subtypes assembled from α4β2/3 with or without the δ subunit. Although the gating of these receptors is subject to dynamic modulation by agents such as anesthetics, barbiturates, and neurosteroids, the cellular mechanisms neurons use to regulate their accumulation on the neuronal plasma membrane remain to be determined. Using immunoprecipitation coupled with metabolic labeling, we demonstrate that the α4 subunit is phosphorylated at Ser(443) by protein kinase C (PKC) in expression systems and hippocampal slices. In addition, the β3 subunit is phosphorylated on serine residues 408/409 by PKC activity, whereas the δ subunit did not appear to be a PKC substrate. We further demonstrate that the PKC-dependent increase of the cell surface expression of α4 subunit-containing GABA(A)Rs is dependent on Ser(443). Mechanistically, phosphorylation of Ser(443) acts to increase the stability of the α4 subunit within the endoplasmic reticulum, thereby increasing the rate of receptor insertion into the plasma membrane. Finally, we show that phosphorylation of Ser(443) increases the activity of α4 subunit-containing GABA(A)Rs by preventing current run-down. These results suggest that PKC-dependent phosphorylation of the α4 subunit plays a significant role in enhancing the cell surface stability and activity of GABA(A)R subtypes that mediate tonic inhibition.

The presence of phosphatidylinositol-4,5-bisphosphate directly impacts on amphetamine-induced serotonin transporter-mediated efflux

Monoaminergic neurotransmitters are released into the synaptic cleft by exocytosis; sodium chloride-dependent monoamine transporters like the serotonin transporter (SERT) efficiently clear the synapse of serotonin to stop neurotransmission. Administration of amphetamines (AMPH), induces the reversal of substrate transport. It has been repeatedly shown that inward and outward transport can be independently regulated; phosphorylation of key residues in the amino-terminus is thought to be the responsible mechanism. In our study, we provide evidence for a novel intracellular regulation mechanism of SERT. Depletion of phosphatidylinositol-4,5-bisphosphate (PIP2) reduced AMPH-induced SERT-mediated substrate release specifically. This effect was limited to SERT-mediated efflux because SERT-mediated reuptake was completely unaffected. PIP2 depletion was achieved by activation of phospholipase C (PLC) upon application of 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide (m-3M3FBS; 25 μM), a direct and potent PLC activator. Efflux was determined in a superfusion system that allows for measurement of AMPH-triggered fractional release rates of substrate efflux. Preincubation (10 min) of SERT wild-type-expressing cells with m-3M3FBS led to a significant and concentration-dependent reduction of AMPH-induced efflux. This effect was largely blocked by co-application of {type:entrez-nucleotide,attrs:{text:U73122,term_id:4098075,term_text:U73122}}U73122 (5 μM; 2 min), a PLC inhibitor. PLC-mediated PIP2 hydrolysation generates inositol-3-phosphate and diacylglycerol and thereby provides downstream effects like protein kinase C (PKC) activation and intracellular calcium rise. Thus we (i) stimulated PKC with PMA (1.0 μM; 10 min), (ii) inhibited with GF109203X (1.0 μM; 10 min) and (iii) chelated Ca2+ with BAPTA-AM (50 μM; 30 min): There was no effect on efflux with or without m-3M3FBS. This strongly supports the notion that the presence of PIP2 in the immediate vicinity of SERT is essential for the AMPH-induced SERT-mediated efflux. HEK cells endogenously express P2Y receptors which activate PLC by Gqα. Stimulation of these receptors with ATP and ADP (under suppression of phosphatidylinositol-4-kinase by 30 μM phenylarsineoxide) showed a reduction of release. The trafficking process of SERT was excluded by fluorescence confocal experiments upon administration of m-3M3FBS. Perforated patch-clamp recordings on hSERT-expressing HEK cells using m-3M3FBS and PCA showed a significant reduction of membrane current effects compared to control. Additionally, these results provide evidence for the necessity of the presence of PIP2 in the plasma membrane for the AMPH-induced outward configuration of SERT.

Phosphoregulation of Twist1 provides a mechanism of cell fate control.

Basic Helix-loop-Helix (bHLH) factors play a significant role in both development and disease. bHLH factors function as protein dimers where two bHLH factors compose an active transcriptional complex. In various species, the bHLH factor Twist has been shown to play critical roles in diverse developmental systems such as mesoderm formation, neurogenesis, myogenesis, and neural crest cell migration and differentiation. Pathologically, Twist1 is a master regulator of epithelial-to-mesenchymal transition (EMT) and is causative of the autosomal-dominant human disease Saethre Chotzen Syndrome (SCS). Given the wide spectrum of Twist1 expression in the developing embryo and the diverse roles it plays within these forming tissues, the question of how Twist1 fills some of these specific roles has been largely unanswered. Recent work has shown that Twist’s biological function can be regulated by its partner choice within a given cell. Our work has identified a phosphoregulatory circuit where phosphorylation of key residues within the bHLH domain alters partner affinities for Twist1; and more recently, we show that the DNA binding affinity of the complexes that do form is affected in a cis-element dependent manner. Such perturbations are complex as they not only affect direct transcriptional programs of Twist1, but they indirectly affect the transcriptional outcomes of any bHLH factor that can dimerize with Twist1. Thus, the resulting lineage-restricted cell fate defects are a combination of loss-of-function and gain-of-function events. Relating the observed phenotypes of defective Twist function with this complex regulatory mechanism will add insight into our understanding of the critical functions of this complex transcription factor.

Dissociation of endothelial nitric oxide synthase phosphorylation and activity in uterine artery endothelial cells

Pregnancy enhanced nitric oxide production by uterine artery endothelial cells (UAEC) is the result of reprogramming of both Ca(2+) and kinase signaling pathways. Using UAEC derived from pregnant ewes (P-UAEC), as well as COS-7 cells transiently expressing ovine endothelial nitric oxide synthase (eNOS), we investigated the role of phosphorylation of five known amino acids following treatment with physiological calcium-mobilizing agent ATP and compared with the effects of PMA (also known as TPA) alone or in combination with ATP. In P-UAEC, ATP stimulated eNOS activity and phosphorylation of eNOS S617, S635, and S1179. PMA promoted eNOS phosphorylation but without activation. PMA and ATP cotreatment attenuated ATP-stimulated activity despite no increase in phospho (p)-T497 and potentiation of p-S1179. In COS-7 cells, PMA inhibition of ATP-stimulated eNOS activity was associated with p-T497 phosphorylation. Although T497D eNOS activity was reduced to 19% of wild-type eNOS with ATP and 44% with A23187, we nonetheless observed more p-S1179 with ATP than with A23187 (3.4-fold and 1.8-fold of control, respectively). Furthermore, the S1179A eNOS mutation partly attenuated ATP- but not A23187-stimulated activity, but when combined with T497D, no further reduction of eNOS activity was observed. In conclusion, although phosphorylation of eNOS is associated with activation in P-UAEC, no single or combination of phosphorylation events predict activity changes. In COS-7 cells, phosphorylation of T497 can attenuate activity but also influences S1179 phosphorylation. We conclude that in both cell types, observed changes in phosphorylation of key residues may influence eNOS activation but are not sufficient alone to describe eNOS activation.