Phosphorylation Of Par-4 Research Articles

GRK6 regulates the hemostatic response to injury through its rate-limiting effects on GPCR signaling in platelets

AbstractG protein–coupled receptors (GPCRs) mediate the majority of platelet activation in response to agonists. However, questions remain regarding the mechanisms that provide negative feedback toward activated GPCRs to limit platelet activation and thrombus formation. Here we provide the first evidence that GPCR kinase 6 (GRK6) serves this role in platelets, using GRK6−/− mice generated by CRISPR-Cas9 genome editing to examine the consequences of GRK6 knockout on GPCR-dependent signaling. Hemostatic thrombi formed in GRK6−/− mice are larger than in wild-type (WT) controls during the early stages of thrombus formation, with a rapid increase in platelet accumulation at the site of injury. GRK6−/− platelets have increased platelet activation, but in an agonist-selective manner. Responses to PAR4 agonist or adenosine 5′-diphosphate stimulation in GRK6−/− platelets are increased compared with WT littermates, whereas the response to thromboxane A2 (TxA2) is normal. Underlying these changes in GRK6−/− platelets is an increase in Ca2+ mobilization, Akt activation, and granule secretion. Furthermore, deletion of GRK6 in human MEG-01 cells causes an increase in Ca2+ response and PAR1 surface expression in response to thrombin. Finally, we show that human platelet activation in response to thrombin causes an increase in binding of GRK6 to PAR1, as well as an increase in the phosphorylation of PAR1. Deletion of GRK6 in MEG-01 cells causes a decrease in PAR1 phosphorylation. Taken together, these data show that GRK6 regulates the hemostatic response to injury through PAR- and P2Y12-mediated effects, helping to limit the rate of platelet activation during thrombus growth and prevent inappropriate platelet activation.

BMX Represses Thrombin-PAR1-Mediated Endothelial Permeability and Vascular Leakage During Early Sepsis.

BMX (bone marrow kinase on the X chromosome) is highly expressed in the arterial endothelium from the embryonic stage to the adult stage in mice. It is also expressed in microvessels and the lymphatics in response to pathological stimuli. However, its role in endothelial permeability and sepsis remains unknown. We aimed to delineate the function of BMX in thrombin-mediated endothelial permeability and the vascular leakage that occurs with sepsis in cecal ligation and puncture models. The cecal ligation and puncture model was applied to WT (wild type) and BMX-KO (BMX global knockout) mice to induce sepsis. Meanwhile, the electric cell-substrate impedance sensing assay was used to detect transendothelial electrical resistance in vitro and, the modified Miles assay was used to evaluate vascular leakage in vivo. We showed that BMX loss caused lung injury and inflammation in early cecal ligation and puncture-induced sepsis. Disruption of BMX increased thrombin-mediated permeability in mice and cultured endothelial cells by 2- to 3-fold. The expression of BMX in macrophages, neutrophils, platelets, and lung epithelial cells was undetectable compared with that in endothelial cells, indicating that endothelium dysfunction, rather than leukocyte and platelet dysfunction, was involved in vascular permeability and sepsis. Mechanistically, biochemical and cellular analyses demonstrated that BMX specifically repressed thrombin-PAR1 (protease-activated receptor-1) signaling in endothelial cells by directly phosphorylating PAR1 and promoting its internalization and deactivation. Importantly, pretreatment with the selective PAR1 antagonist SCH79797 rescued BMX loss-mediated endothelial permeability and pulmonary leakage in early cecal ligation and puncture-induced sepsis. Acting as a negative regulator of PAR1, BMX promotes PAR1 internalization and signal inactivation through PAR1 phosphorylation. Moreover, BMX-mediated PAR1 internalization attenuates endothelial permeability to protect vascular leakage during early sepsis.

Rho-associated kinase and zipper-interacting protein kinase, but not myosin light chain kinase, are involved in the regulation of myosin phosphorylation in serum-stimulated human arterial smooth muscle cells.

Myosin regulatory light chain (LC20) phosphorylation plays an important role in vascular smooth muscle contraction and cell migration. Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) phosphorylates LC20 (its only known substrate) exclusively at S19. Rho-associated kinase (ROCK) and zipper-interacting protein kinase (ZIPK) have been implicated in the regulation of LC20 phosphorylation via direct phosphorylation of LC20 at T18 and S19 and indirectly via phosphorylation of MYPT1 (the myosin targeting subunit of myosin light chain phosphatase, MLCP) and Par-4 (prostate-apoptosis response-4). Phosphorylation of MYPT1 at T696 and T853 inhibits MLCP activity whereas phosphorylation of Par-4 at T163 disrupts its interaction with MYPT1, exposing the sites of phosphorylation in MYPT1 and leading to MLCP inhibition. To evaluate the roles of MLCK, ROCK and ZIPK in these phosphorylation events, we investigated the time courses of phosphorylation of LC20, MYPT1 and Par-4 in serum-stimulated human vascular smooth muscle cells (from coronary and umbilical arteries), and examined the effects of siRNA-mediated MLCK, ROCK and ZIPK knockdown and pharmacological inhibition on these phosphorylation events. Serum stimulation induced rapid phosphorylation of LC20 at T18 and S19, MYPT1 at T696 and T853, and Par-4 at T163, peaking within 30–120 s. MLCK knockdown or inhibition, or Ca2+ chelation with EGTA, had no effect on serum-induced LC20 phosphorylation. ROCK knockdown decreased the levels of phosphorylation of LC20 at T18 and S19, of MYPT1 at T696 and T853, and of Par-4 at T163, whereas ZIPK knockdown decreased LC20 diphosphorylation, but increased phosphorylation of MYPT1 at T696 and T853 and of Par-4 at T163. ROCK inhibition with GSK429286A reduced serum-induced phosphorylation of LC20 at T18 and S19, MYPT1 at T853 and Par-4 at T163, while ZIPK inhibition by HS38 reduced only LC20 diphosphorylation. We also demonstrated that serum stimulation induced phosphorylation (activation) of ZIPK, which was inhibited by ROCK and ZIPK down-regulation and inhibition. Finally, basal phosphorylation of LC20 in the absence of serum stimulation was unaffected by MLCK, ROCK or ZIPK knockdown or inhibition. We conclude that: (i) serum stimulation of cultured human arterial smooth muscle cells results in rapid phosphorylation of LC20, MYPT1, Par-4 and ZIPK, in contrast to the slower phosphorylation of kinases and other proteins involved in other signaling pathways (Akt, ERK1/2, p38 MAPK and HSP27), (ii) ROCK and ZIPK, but not MLCK, are involved in serum-induced phosphorylation of LC20, (iii) ROCK, but not ZIPK, directly phosphorylates MYPT1 at T853 and Par-4 at T163 in response to serum stimulation, (iv) ZIPK phosphorylation is enhanced by serum stimulation and involves phosphorylation by ROCK and autophosphorylation, and (v) basal phosphorylation of LC20 under serum-free conditions is not attributable to MLCK, ROCK or ZIPK.

GRK6 Regulates the Hemostatic Response to Injury through Its Rate-Limiting Effects on GPCR Signaling in Platelets

Inappropriate platelet activation remains a major cause of cardiovascular and cerebrovascular diseases. Most agonists activate platelets through G protein-coupled receptors (GPCRs). However, questions remain about mechanisms that provide negative feedback towards activated GPCRs to limit platelet activation and thrombus formation. Here we provide the first evidence that GPCR kinase 6 (GRK6) serves this role in platelets, using GRK6-/- mice generated by CRISPR-Cas9 genome editing to examine the consequences of GRK6 knockout on GPCR-dependent signaling. Hemostatic thrombi formed in GRK6-/- mice are larger than in WT controls during the early stages of thrombus formation, with a rapid increase of platelet accumulation at site of injury. Platelet activation in the absence of GRK6 is enhanced, but in an agonist-selective manner. Responses to PAR4 agonist peptide or ADP stimulation in GRK6-/- platelets are increased compared to WT control littermates, while the response to TxA2 is normal. Underlying these changes in GRK6-/- platelets is an increase in Ca2+ mobilization, Akt activation, and granule secretion. Furthermore, deletion of GRK6 in human MEG-01 cells causes an increase in Ca2+ response and PAR1 surface expression in response to thrombin. Finally, we show that in human platelets, platelet activation in response to thrombin causes an increase in binding of GRK6 to PAR1, as well as an increase of the phosphorylation of PAR1. Deletion of GRK6 in MEG-01 cells causes a decrease in PAR1 phosphorylation. Collectively, these observations, for the first time, show that 1) GRK6 regulates the hemostatic response to injury by thrombin and ADP, 2) it mediates platelet activation by reducing PAR1/4- and P2Y12-dependent signaling, and 3) GRK6 limits the rate of platelet activation during early stage of thrombus growth and helps prevent inappropriate platelet activation. Disclosures No relevant conflicts of interest to declare.

Protein clustering for cell polarity: Par-3 as a paradigm.

The scaffold protein Par-3 ( Drosophila Bazooka) is a central organizer of cell polarity across animals. This review focuses on how the clustering of Par-3 contributes to cell polarity. It begins with the Par-3 homo-oligomerization mechanism and its regulation by Par-1 phosphorylation. The role of polarized cytoskeletal networks in distributing Par-3 clusters to one end of the cell is then discussed, as is the subsequent maintenance of polarized Par-3 clusters through hindered mobility and inhibition from the opposite pole. Finally, specific roles of Par-3 clusters are reviewed, including the bundling of microtubules, the cortical docking of centrosomes, the growth and positioning of cadherin–catenin clusters, and the inhibition of the Par-6–aPKC kinase cassette. Examples are drawn from Drosophila, Caenorhabditis elegans, mammalian cell culture, and biochemical studies.

Gliotactin and Discs large are co-regulated to maintain epithelial integrity

Establishment and maintenance of permeability barriers is one of the most important functions of epithelial cells. Tricellular junctions (TCJs) maintain the permeability barriers at the contact site of three epithelial cells. Gliotactin, a member of the Neuroligin family, is the only known Drosophila protein exclusively localized to the TCJ and is necessary for maintenance of the permeability barrier. Overexpression triggers the spread of Gliotactin away from the TCJ and causes epithelial cells to delaminate, migrate and die. Furthermore, excess Gliotactin at the cell membrane results in an extensive downregulation of Discs large (Dlg) at the septate junctions. The intracellular domain of Gliotactin contains two highly conserved tyrosine residues and a PDZ binding motif. We previously found that phosphorylation of the tyrosine residues is necessary to control the level of Gliotactin at the TCJ. In this study we demonstrate that the phenotypes associated with excess Gliotactin are due to a functional interaction between Gliotactin and Dlg that is dependent on both tyrosine phosphorylation as well as the PDZ binding motif. We further show that elevated levels of Dlg strongly enhance Gliotactin overexpression phenotypes to the point where tissue over-growth is observed. The exhibition of these phenotypes require phosphorylation of Dlg on serine 797, a known Par1 phosphorylation target. Blocking this phosphorylation completely suppresses the cell invasiveness and apoptotic phenotypes associated with Gliotactin overexpression. Additionally, we show that Drosophila JNK acts downstream of Gliotactin and Dlg to mediate the overgrowth and apoptosis caused by the functional interaction of Gliotactin and Dlg.

Prostate-apoptosis response-4 phosphorylation in vascular smooth muscle

The protein prostate-apoptosis response (Par)-4 has been implicated in the regulation of smooth muscle contraction, based largely on studies with the A7r5 cell line. A mechanism has been proposed whereby Par-4 binding to MYPT1 (the myosin-targeting subunit of myosin light chain phosphatase, MLCP) blocks access of zipper-interacting protein kinase (ZIPK) to Thr697 and Thr855 of MYPT1, whose phosphorylation is associated with MLCP inhibition. Phosphorylation of Par-4 at Thr155 disrupts its interaction with MYPT1, exposing the sites of phosphorylation in MYPT1 and leading to MLCP inhibition and contraction. We tested this “padlock” hypothesis in a well-characterized vascular smooth muscle system, the rat caudal artery. Par-4 was retained in Triton-skinned tissue, suggesting a tight association with the contractile machinery, and indeed Par-4 co-immunoprecipitated with MYPT1. Treatment of Triton-skinned tissue with the phosphatase inhibitor microcystin (MC) evoked phosphorylation of Par-4 at Thr155, but did not induce its dissociation from the contractile machinery. Furthermore, analysis of the time courses of MC-induced phosphorylation of MYPT1 and Par-4 revealed that MYPT1 phosphorylation at Thr697 or Thr855 preceded Par-4 phosphorylation. Par-4 phosphorylation was inhibited by the non-selective kinase inhibitor staurosporine, but not by inhibitors of ZIPK, Rho-associated kinase or protein kinase C. In addition, Par-4 phosphorylation did not occur upon addition of constitutively-active ZIPK to skinned tissue. We conclude that phosphorylation of Par-4 does not regulate contraction of this vascular smooth muscle tissue by inducing dissociation of Par-4 from MYPT1 to allow phosphorylation of MYPT1 and inhibition of MLCP.

The pro-apoptotic protein Prostate Apoptosis Response Protein-4 (Par-4) can be activated in colon cancer cells by treatment with Src inhibitor and 5-FU

The overexpression of the pro-apoptotic protein Prostate Apoptosis Response Protein-4 in colon cancer has been shown to increase response to the chemotherapeutic agent 5-fluorouracil (5-FU). Although colon cancer cells endogenously express Par-4, the presence or overexpression of Par-4 alone does not cause apoptosis. We hypothesize that Par-4 is inactivated in colon cancer. In colon cancer, the levels and the kinase activity of the nonreceptor tyrosine kinase c-Src increase with tumor progression. One of the downstream effectors of c-Src is Akt1. Akt1 has been shown to inhibit the pro-apoptotic activity of Par-4 in prostate cancer cells. We therefore investigated the potential of activating Par-4 by inhibiting c-Src. Colon carcinoma cell lines were treated with the Src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d]pyrimidine (PP2) in combination with the chemotherapeutic agent 5-FU. Treating cells with PP2 and 5-FU resulted in reduced interaction of Par-4 with Akt1 and with the scaffolding protein 14-3-3σ, and mobilization of Par-4 to the nucleus. Par-4 was shown to interact not only with Akt1 and 14-3-3σ, but also with c-Src. Overexpression of c-Src induced the phosphorylation of Par-4 at tyrosine site/s. Thus, in this study, we have shown that Par-4 can be activated by inhibiting Src with a pharmacological inhibitor and adding a chemotherapeutic agent. The activation of the pro-apoptotic protein Par-4 as reported in this study is a novel mechanism by which apoptosis occurs with a Src kinase inhibitor and 5-FU. In addition, we have demonstrated that the pro-apoptotic activity of endogenously expressed Par-4 can be increased in colon cancer cells.

Arrestin-2 Differentially Regulates PAR4 and ADP Receptor Signaling in Platelets

Arrestins can facilitate desensitization or signaling by G protein-coupled receptors (GPCR) in many cells, but their roles in platelets remain uncharacterized. Because of recent reports that arrestins can serve as scaffolds to recruit phosphatidylinositol-3 kinases (PI3K)s to GPCRs, we sought to determine whether arrestins regulate PI3K-dependent Akt signaling in platelets, with consequences for thrombosis. Co-immunoprecipitation experiments demonstrate that arrestin-2 associates with p85 PI3Kα/β subunits in thrombin-stimulated platelets, but not resting cells. The association is inhibited by inhibitors of P2Y12 and Src family kinases (SFKs). The function of arrestin-2 in platelets is agonist-specific, as PAR4-dependent Akt phosphorylation and fibrinogen binding were reduced in arrestin-2 knock-out platelets compared with WT controls, but ADP-stimulated signaling to Akt and fibrinogen binding were unaffected. ADP receptors regulate arrestin recruitment to PAR4, because co-immunoprecipitates of arrestin-2 with PAR4 are disrupted by inhibitors of P2Y1 or P2Y12. P2Y1 may regulate arrestin-2 recruitment to PAR4 through protein kinase C (PKC) activation, whereas P2Y12 directly interacts with PAR4 and therefore, may help to recruit arrestin-2 to PAR4. Finally, arrestin2(-/-) mice are less sensitive to ferric chloride-induced thrombosis than WT mice, suggesting that arrestin-2 can regulate thrombus formation in vivo. In conclusion, arrestin-2 regulates PAR4-dependent signaling pathways, but not responses to ADP alone, and contributes to thrombus formation in vivo.

Par-4 Is an Essential Downstream Target of DAP-like Kinase (Dlk) in Dlk/Par-4–mediated Apoptosis

Prostate apoptosis response-4 (Par-4) was initially identified as a gene product up-regulated in prostate cancer cells undergoing apoptosis. In rat fibroblasts, coexpression of Par-4 and its interaction partner DAP-like kinase (Dlk, which is also known as zipper-interacting protein kinase [ZIPK]) induces relocation of the kinase from the nucleus to the actin filament system, followed by extensive myosin light chain (MLC) phosphorylation and induction of apoptosis. Our analyses show that the synergistic proapoptotic effect of Dlk/Par-4 complexes is abrogated when either Dlk/Par-4 interaction or Dlk kinase activity is impaired. In vitro phosphorylation assays employing Dlk and Par-4 phosphorylation mutants carrying alanine substitutions for residues S154, T155, S220, or S249, respectively, identified T155 as the major Par-4 phosphorylation site of Dlk. Coexpression experiments in REF52.2 cells revealed that phosphorylation of Par-4 at T155 by Dlk was essential for apoptosis induction in vivo. In the presence of the Par-4 T155A mutant Dlk was partially recruited to actin filaments but resided mainly in the nucleus. Consequently, apoptosis was not induced in Dlk/Par-4 T155A-expressing cells. In vivo phosphorylation of Par-4 at T155 was demonstrated with a phospho-specific Par-4 antibody. Our results demonstrate that Dlk-mediated phosphorylation of Par-4 at T155 is a crucial event in Dlk/Par-4-induced apoptosis.

Helicobacter pylori CagA targets PAR1/MARK kinase to disrupt epithelial cell polarity

Helicobacter pylori cagA-positive strains are associated with gastritis, ulcerations and gastric adenocarcinoma. CagA is delivered into gastric epithelial cells and, on tyrosine phosphorylation, specifically binds and activates the SHP2 oncoprotein, thereby inducing the formation of an elongated cell shape known as the 'hummingbird' phenotype. In polarized epithelial cells, CagA also disrupts the tight junction and causes loss of apical-basolateral polarity. We show here that H. pylori CagA specifically interacts with PAR1/MARK kinase, which has an essential role in epithelial cell polarity. Association of CagA inhibits PAR1 kinase activity and prevents atypical protein kinase C (aPKC)-mediated PAR1 phosphorylation, which dissociates PAR1 from the membrane, collectively causing junctional and polarity defects. Because of the multimeric nature of PAR1 (ref. 14), PAR1 also promotes CagA multimerization, which stabilizes the CagA-SHP2 interaction. Furthermore, induction of the hummingbird phenotype by CagA-activated SHP2 requires simultaneous inhibition of PAR1 kinase activity by CagA. Thus, the CagA-PAR1 interaction not only elicits the junctional and polarity defects but also promotes the morphogenetic activity of CagA. Our findings revealed that PAR1 is a key target of H. pylori CagA in the disorganization of gastric epithelial architecture underlying mucosal damage, inflammation and carcinogenesis.

Antagonistic functions of Par-1 kinase and protein phosphatase 2A are required for localization of Bazooka and photoreceptor morphogenesis in Drosophila

Establishment and maintenance of apical basal cell polarity are essential for epithelial morphogenesis and have been studied extensively using the Drosophila eye as a model system. Bazooka (Baz), a component of the Par-6 complex, plays important roles in cell polarity in diverse cell types including the photoreceptor cells. In ovarian follicle cells, localization of Baz at the apical region is regulated by Par-1 protein kinase. In contrast, Baz in photoreceptor cells is targeted to adherens junctions (AJs). To examine the regulatory pathways responsible for Baz localization in photoreceptor cells, we studied the effects of Par-1 on Baz localization in the pupal retina. Loss of Par-1 impairs the maintenance of AJ markers including Baz and apical polarity proteins of photoreceptor cells but not the establishment of cell polarity. In contrast, overexpression of Par-1 or Baz causes severe mislocalization of junctional and apical markers, resulting in abnormal cell polarity. However, flies with similar overexpression of kinase-inactive mutant Par-1 or unphosphorylatable mutant Baz protein show relatively normal photoreceptor development. These results suggest that dephosphorylation of Baz at the Par-1 phosphorylation sites is essential for proper Baz localization. We also show that the inhibition of protein phosphatase 2A (PP2A) mimics the polarity defects caused by Par-1 overexpression. Furthermore, Par-1 gain-of-function phenotypes are strongly enhanced by reduced PP2A function. Thus, we propose that antagonism between PP2A and Par-1 plays a key role in Baz localization at AJ in photoreceptor morphogenesis.

Binding and Phosphorylation of Par-4 by Akt Is Essential for Cancer Cell Survival

Activation of the PI3K-Akt pathway by loss of tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) function, increased growth factor signaling, or oncogene expression renders cancer cells resistant to apoptotic signals and promotes tumor growth. Although Akt acts as a global survival signal, the molecular circuits of this pathway have not been completely established. We report that Akt physically binds to the pro-apoptotic protein Par-4 via the Par-4 leucine zipper domain and phosphorylates Par-4 to inhibit apoptosis. Suppression of Akt activation by the PI3K-inhibitor PTEN or LY294002, Akt expression by RNA-interference, or Akt function by dominant-negative Akt caused apoptosis in cancer cells. Apoptosis induced by inhibiting Akt was blocked by inhibition of Par-4 expression, but not by inhibition of other apoptosis agonists that are Akt substrates, suggesting that inhibition of the PI3K-Akt pathway leads to Par-4-dependent apoptosis. Thus, Par-4 is essential for PTEN-inducible apoptosis, and inactivation of Par-4 by Akt promotes cancer cell survival.

Distinct PAR-1 Proteins Function in Different Branches of Wnt Signaling during Vertebrate Development

The kinase PAR-1 plays conserved roles in cell polarity. PAR-1 has also been implicated in axis establishment in C. elegans and Drosophila and in Wnt signaling, but its role in vertebrate development is unclear. Here we report that PAR-1 has two distinct and essential roles in axial development in Xenopus mediated by different PAR-1 isoforms. Depletion of PAR-1A or PAR-1BX causes dorsoanterior deficits, reduced Spemann organizer gene expression, and inhibition of canonical Wnt-beta-catenin signaling. By contrast, PAR-1BY depletion inhibits cell movements and localization of Dishevelled protein to the cell cortex, processes associated with noncanonical Wnt signaling. PAR-1 phosphorylation sites in Dishevelled are required for this translocation, but not for canonical Wnt signaling. We conclude that PAR-1BY is required in the PCP branch and mediates Dsh membrane localization while PAR-1A and PAR-1BX are essential for canonical signaling to beta-catenin, possibly via targets other than Dishevelled.

Phosphorylation of Par-4 by Protein Kinase A Is Critical for Apoptosis

Despite distinct dissimilarities, diverse cancers express several common protumorigenic traits. We present here evidence that the proapoptotic protein Par-4 utilizes one such common tumorigenic trait to become selectively activated and induce apoptosis in cancer cells. Elevated protein kinase A (PKA) activity noted in cancer cells activated the apoptotic function of ectopic Par-4 or its SAC (selective for apoptosis induction in cancer cells) domain, which induces apoptosis selectively in cancer cells and not in normal or immortalized cells. PKA preferentially phosphorylated Par-4 at the T155 residue within the SAC domain in cancer cells. Moreover, pharmacological-, peptide-, or small interfering RNA-mediated inhibition of PKA activity in cancer cells resulted in abrogation of both T155 phosphorylation and apoptosis by Par-4. The mechanism of activation of endogenous Par-4 was similar to that of ectopic Par-4, and in response to exogenous stimuli, endogenous Par-4 induced apoptosis by a PKA- and phosphorylated T155-dependent mechanism. Enforced elevation of PKA activity in normal cells resulted in apoptosis by the SAC domain of Par-4 in a T155-dependent manner. Together, these observations suggest that selective apoptosis of cancer cells by the SAC domain of Par-4 involves phosphorylation of T155 by PKA. These findings uncover a novel mechanism engaging PKA, a procancerous activity commonly elevated in most tumor cells, to activate the cancer selective apoptotic action of Par-4.

PAR-1 Kinase Plays an Initiator Role in a Temporally Ordered Phosphorylation Process that Confers Tau Toxicity in Drosophila

Multisite hyperphosphorylation of tau has been implicated in the pathogenesis of neurodegenerative diseases including Alzheimer's disease (AD). However, the phosphorylation events critical for tau toxicity and mechanisms regulating these events are largely unknown. Here we show that Drosophila PAR-1 kinase initiates tau toxicity by triggering a temporally ordered phosphorylation process. PAR-1 directly phosphorylates tau at S262 and S356. This phosphorylation event is a prerequisite for the action of downstream kinases, including glycogen synthase kinase 3 (GSK-3) and cyclin-dependent kinase-5 (Cdk5), to phosphorylate several other sites and generate disease-associated phospho-epitopes. The initiator role of PAR-1 is further underscored by the fact that mutating PAR-1 phosphorylation sites causes a much greater reduction of overall tau phosphorylation and toxicity than mutating S202, one of the downstream sites whose phosphorylation depends on prior PAR-1 action. These findings begin to differentiate the effects of various phosphorylation events on tau toxicity and provide potential therapeutic targets.

Protease-activated Receptors 1 and 4 Are Shut Off with Distinct Kinetics after Activation by Thrombin

Protease-activated receptors 1 and 4 (PAR1 and PAR4) mediate thrombin signaling in human platelets. Whether these receptors are redundant, interact, or serve only partially overlapping functions is unknown. We report that PAR1 and PAR4 signal with distinct tempos. In transfected fibroblasts, PAR4 triggered substantially more phosphoinositide hydrolysis per activated receptor than PAR1 and was shut off more slowly than PAR1. Shutoff and internalization of PAR1 depends upon phosphorylation of its carboxyl tail upon receptor activation. In contrast to PAR1, phosphorylation of PAR4 was undetectable, and activation-dependent internalization of PAR4 was much slower than that seen for PAR1. Mutation of potential phosphorylation sites in the carboxyl tail of PAR1 enhanced PAR1 signaling, whereas analogous mutations in PAR4 had no effect. Thus PAR4 signaling is shut off less rapidly than PAR1, probably due to differences in receptor phosphorylation. PAR1 and PAR4 also signaled with distinct tempos in platelets. PAR1 triggered a rapid and transient increase in intracellular calcium, whereas PAR4 triggered a more prolonged response. Together, the tempo of these responses accounted for that triggered by thrombin. Thus differences in the rates at which PAR1 and PAR4 are shut off allow thrombin to trigger intracellular signaling with distinct temporal characteristics.