The Methyltransferase WBSCR22/Merm1 Enhances Glucocorticoid Receptor Function and Is Regulated in Lung Inflammation and Cancer

Glucocorticoids (GC) regulate cell fate and immune function. We identified the metastasis-promoting methyltransferase, metastasis-related methyltransferase 1 (WBSCR22/Merm1) as a novel glucocorticoid receptor (GR) regulator relevant to human disease. Merm1 binds the GR co-activator GRIP1 but not GR. Loss of Merm1 impaired both GR transactivation and transrepression by reducing GR recruitment to its binding sites. This was accompanied by loss of GR-dependent H3K4Me3 at a well characterized promoter. Inflammation promotes GC resistance, in part through the actions of TNFα and IFNγ. These cytokines suppressed Merm1 protein expression by driving ubiquitination of two conserved lysine residues. Restoration of Merm1 expression rescued GR transactivation. Cytokine suppression of Merm1 and of GR function was also seen in human lung explants. In addition, striking loss of Merm1 protein was observed in both inflammatory and neoplastic human lung pathologies. In conclusion, Merm1 is a novel regulator of chromatin structure affecting GR recruitment and function, contributing to loss of GC sensitivity in inflammation, with suppressed expression in pulmonary disease.     

Post-phosphorylation prolyl isomerisation of gephyrin represents a mechanism to modulate glycine receptors function

The microtubule binding protein gephyrin plays a prominent role in establishing and maintaining a high concentration of inhibitory glycine receptors juxtaposed to presynaptic releasing sites. Here, we show that endogenous gephyrin undergoes proline-directed phosphorylation, which is followed by the recruitment of the peptidyl-prolyl isomerase Pin1. The interaction between gephyrin and Pin1 is strictly dependent on gephyrin phosphorylation and requires serine-proline consensus sites encompassing the gephyrin proline-rich domain. Upon binding, Pin1 triggers conformational changes in the gephyrin molecule, thus enhancing its ability to bind the beta subunit of GlyRs. Consistently, a downregulation of GlyR clusters was detected in hippocampal neurons derived from Pin1 knockout mice, which was paralleled by a reduction in the amplitude of glycine-evoked currents. Our results suggest that phosphorylation-dependent prolyl isomerisation of gephyrin represents a mechanism for regulating GlyRs function.     

Isomerase Pin1 Stimulates Dephosphorylation of Tau Protein at Cyclin-dependent Kinase (Cdk5)-dependent Alzheimer Phosphorylation Sites

Neurodegenerative diseases associated with the pathological aggregation of microtubule-associated protein Tau are classified as tauopathies. Alzheimer disease, the most common tauopathy, is characterized by neurofibrillary tangles that are mainly composed of abnormally phosphorylated Tau. Similar hyperphosphorylated Tau lesions are found in patients with frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) that is induced by mutations within the tau gene. To further understand the etiology of tauopathies, it will be important to elucidate the mechanism underlying Tau hyperphosphorylation. Tau phosphorylation occurs mainly at proline-directed Ser/Thr sites, which are targeted by protein kinases such as GSK3β and Cdk5. We reported previously that dephosphorylation of Tau at Cdk5-mediated sites was enhanced by Pin1, a peptidyl-prolyl isomerase that stimulates dephosphorylation at proline-directed sites by protein phosphatase 2A. Pin1 deficiency is suggested to cause Tau hyperphosphorylation in Alzheimer disease. Up to the present, Pin1 binding was only shown for two Tau phosphorylation sites (Thr-212 and Thr-231) despite the presence of many more hyperphosphorylated sites. Here, we analyzed the interaction of Pin1 with Tau phosphorylated by Cdk5-p25 using a GST pulldown assay and Biacore approach. We found that Pin1 binds and stimulates dephosphorylation of Tau at all Cdk5-mediated sites (Ser-202, Thr-205, Ser-235, and Ser-404). Furthermore, FTDP-17 mutant Tau (P301L or R406W) showed slightly weaker Pin1 binding than non-mutated Tau, suggesting that FTDP-17 mutations induce hyperphosphorylation by reducing the interaction between Pin1 and Tau. Together, these results indicate that Pin1 is generally involved in the regulation of Tau hyperphosphorylation and hence the etiology of tauopathies.     

Inhibition of Pin1 reduces glutamate-induced perikaryal accumulation of phosphorylated neurofilament-H in neurons

Under normal conditions, the proline-directed serine/threonine residues of neurofilament tail-domain repeats are exclusively phosphorylated in axons. In pathological conditions such as amyotrophic lateral sclerosis (ALS), motor neurons contain abnormal perikaryal accumulations of phosphorylated neurofilament proteins. The precise mechanisms for this compartment-specific phosphorylation of neurofilaments are not completely understood. Although localization of kinases and phosphatases is certainly implicated, another possibility involves Pin1 modulation of phosphorylation of the proline-directed serine/threonine residues. Pin1, a prolyl isomerase, selectively binds to phosphorylated proline-directed serine/threonine residues in target proteins and isomerizes cis isomers to more stable trans configurations. In this study we show that Pin1 associates with phosphorylated neurofilament-H (p-NF-H) in neurons and is colocalized in ALS-affected spinal cord neuronal inclusions. To mimic the pathology of neurodegeneration, we studied glutamate-stressed neurons that displayed increased p-NF-H in perikaryal accumulations that colocalized with Pin1 and led to cell death. Both effects were reduced upon inhibition of Pin1 activity by the use of an inhibitor juglone and down-regulating Pin1 levels through the use of Pin1 small interfering RNA. Thus, isomerization of lys-ser-pro repeat residues that are abundant in NF-H tail domains by Pin1 can regulate NF-H phosphorylation, which suggests that Pin1 inhibition may be an attractive therapeutic target to reduce pathological accumulations of p-NF-H.     

The Cellular Peptidyl-Prolyl cis/trans Isomerase Pin1 Regulates Reactivation of Kaposi's Sarcoma-Associated Herpesvirus from Latency

Kaposi''s sarcoma-associated herpesvirus (KSHV) causes Kaposi''s sarcoma and primary effusion lymphoma. KSHV-infected cells are predominantly latent, with a subset undergoing lytic reactivation. Rta is the essential lytic switch protein that reactivates virus by forming transactivation-competent complexes with the Notch effector protein RBP-Jk and promoter DNA. Strikingly, Rta homolog analysis reveals that prolines constitute 17% of conserved residues. Rta is also highly phosphorylated in vivo. We previously demonstrated that proline content determines Rta homotetramerization and function. We hypothesize that proline-directed modifications regulate Rta function by controlling binding to peptidyl-prolyl cis/trans isomerases (PPIases). Cellular PPIase Pin1 binds specifically to phosphoserine- or phosphothreonine-proline (pS/T-P) motifs in target proteins. Pin1 dysregulation is implicated in myriad human cancers and can be subverted by viruses. Our data show that KSHV Rta protein contains potential pS/T-P motifs and binds directly to Pin1. Rta transactivation is enhanced by Pin1 at two delayed early viral promoters in uninfected cells. Pin1''s effect, however, suggests a rheostat-like influence on Rta function. We show that in infected cells, endogenous Pin1 is active during reactivation and enhances Rta-dependent early protein expression induced by multiple signals, as well as DNA replication. Surprisingly, ablation of Pin1 activity by the chemical juglone or dominant-negative Pin1 enhanced late gene expression and production of infectious virus, while ectopic Pin1 showed inhibitory effects. Our data thus suggest that Pin1 is a unique, dose-dependent molecular timer that enhances Rta protein function, but inhibits late gene synthesis and virion production, during KSHV lytic reactivation.     

γ-Aminobutyric acid type A (GABAA) receptor activation modulates tau phosphorylation

Abnormal phosphorylation and aggregation of the microtubule-associated protein Tau are hallmarks of various neurodegenerative diseases, such as Alzheimer disease. Molecular mechanisms that regulate Tau phosphorylation are complex and currently incompletely understood. We have developed a novel live cell reporter system based on protein-fragment complementation assay to study dynamic changes in Tau phosphorylation status. In this assay, fusion proteins of Tau and Pin1 (peptidyl-prolyl cis-trans-isomerase 1) carrying complementary fragments of a luciferase protein serve as a sensor of altered protein-protein interaction between Tau and Pin1, a critical regulator of Tau dephosphorylation at several disease-associated proline-directed phosphorylation sites. Using this system, we identified several structurally distinct GABA(A) receptor modulators as novel regulators of Tau phosphorylation in a chemical library screen. GABA(A) receptor activation promoted specific phosphorylation of Tau at the AT8 epitope (Ser-199/Ser-202/Thr-205) in cultures of mature cortical neurons. Increased Tau phosphorylation by GABA(A) receptor activity was associated with reduced Tau binding to protein phosphatase 2A and was dependent on Cdk5 but not GSK3β kinase activity.     

Par14 Protein Associates with Insulin Receptor Substrate 1 (IRS-1), Thereby Enhancing Insulin-induced IRS-1 Phosphorylation and Metabolic Actions

Pin1 and Par14 are parvulin-type peptidyl-prolyl cis/trans isomerases. Although numerous proteins have been identified as Pin1 substrates, the target proteins of Par14 remain largely unknown. Par14 expression levels are increased in the livers and embryonic fibroblasts of Pin1 KO mice, suggesting a compensatory relationship between the functions of Pin1 and Par14. In this study, the association of Par14 with insulin receptor substrate 1 (IRS-1) was demonstrated in HepG2 cells overexpressing both as well as endogenously in the mouse liver. The analysis using deletion-mutated Par14 and IRS-1 constructs revealed the N-terminal portion containing the basic domain of Par14 and the two relatively C-terminal portions of IRS-1 to be involved in these associations, in contrast to the WW domain of Pin1 and the SAIN domain of IRS-1. Par14 overexpression in HepG2 markedly enhanced insulin-induced IRS-1 phosphorylation and its downstream events, PI3K binding with IRS-1 and Akt phosphorylation. In contrast, treating HepG2 cells with Par14 siRNA suppressed these events. In addition, overexpression of Par14 in the insulin-resistant ob/ob mouse liver by adenoviral transfer significantly improved hyperglycemia with normalization of hepatic PEPCK and G6Pase mRNA levels, and gene suppression of Par14 using shRNA adenovirus significantly exacerbated the glucose intolerance in Pin1 KO mice. Therefore, although Pin1 and Par14 associate with different portions of IRS-1, the prolyl cis/trans isomerization in multiple sites of IRS-1 by these isomerases appears to be critical for efficient insulin receptor-induced IRS-1 phosphorylation. This process is likely to be one of the major mechanisms regulating insulin sensitivity and also constitutes a potential therapeutic target for novel insulin-sensitizing agents.     

Pin1 interacts with c-Myb in a phosphorylation-dependent manner and regulates its transactivation activity

Activity and stability of the proto-oncogene c-Myb are regulated by post-translational modifications, though the molecular mechanisms underlying such control are only partially understood. Here we describe the functional interaction of c-Myb with Pin1, an isomerase that binds to phosphorylated Ser/Thr-Pro motifs. We found that co-expression of c-Myb and Pin1 led to a net increase of c-Myb transactivation activity, both on reporter constructs as well as on an endogenous target gene. DNA-binding studies revealed that Pin1 did not increase the association of c-Myb with its response element in DNA. The increase of c-Myb transactivation activity was strictly dependent on the presence of an active catalytic center in Pin1. We provide evidence that c-Myb and Pin1 physically interacted, both upon ectopic expression of the proteins in HEK-293 cells as well as in the more physiological setting of HL60 cells, where c-Myb and Pin1 are resident proteins. By point mutating each individual Ser/Thr-Pro motif in c-Myb as well as by using deletion mutants we show that S528 in the EVES-motif was the docking site for Pin1. Mass spectrometry confirmed that S528 is phosphorylated in vivo. Finally, functional studies showed that mutation of S528 to alanine almost abolished the increase of transactivation activity by Pin1. This study reveals a new paradigm by which phosphorylation controls c-Myb function.     

Exceptional disfavor for proline at the P + 1 position among AGC and CAMK kinases establishes reciprocal specificity between them and the proline-directed kinases

To precisely regulate critical signaling pathways, two kinases that phosphorylate distinct sites on the same protein substrate must have mutually exclusive specificity. Evolution could assure this by designing families of kinase such as basophilic kinases and proline-directed kinase with distinct peptide specificity; their reciprocal peptide specificity would have to be very complete, since recruitment of substrate allows phosphorylation of even rather poor phosphorylation sites in a protein. Here we report a powerful evolutionary strategy that assures distinct substrates for basophilic kinases (PKA, PKG and PKC (AGC) and calmodulin-dependent protein kinase (CAMK)) and proline-directed kinase, namely by the presence or absence of proline at the P + 1 position in substrates. Analysis of degenerate and non-degenerate peptides by in vitro kinase assays reveals that proline at the P + 1 position in substrates functions as a "veto" residue in substrate recognition by AGC and CAMK kinases. Furthermore, analysis of reported substrates of two typical basophilic kinases, protein kinase C and protein kinase A, shows the lowest occurrence of proline at the P + 1 position. Analysis of crystal structures and sequence conservation provides a molecular basis for this disfavor and illustrate its generality.     

Phosphorylation-mediated regulation of a rice ABA responsive element binding factor

OREB1 is a rice ABRE binding factor characterized by the presence of multiple highly-conserved phosphorylation domains (C1, C2, C3, and C4) and two kinase recognition motifs, RXXS/T and S/TXXE/D, within different functional domains. An in vitro kinase assay showed that OREB1 is phosphorylated not only by the SnRK2 kinase, but also by other Ser/Thr protein kinases, such as CaMKII, CKII, and SnRK3. Furthermore, the N-terminal phosphorylation domain C1 was found to be differentially phosphorylated by the SnRK2/SnRK3 kinase and by hyperosmotic/cold stress, suggesting that the C1 domain may function in decoding different signals. The phosphorylation-mediated regulation of OREB1 activity was investigated through mutation of the SnRK2 recognition motif RXXS/T within each phosphorylation module. OREB1 contains a crucial nine-amino acid transactivation domain located near the phosphorylation module C1. Deletion of the C1 domain increased OREB1 activity, whereas mutation of Ser 44, Ser 45, and Ser 48 of the C1 domain to aspartates decreased OREB1 activity. In the C2 domain, a double mutation of Ser 118 and Ser 120 to alanines suppressed OREB1 activity. These findings strongly suggest that selective phosphorylation of the C1 or C2 modules may positively or negatively regulate OREB1 transactivation. In addition, mutation of Ser 385 of the C4 domain to alanines completely abolished the interaction between OREB1 and a rice 14-3-3 protein, GF14d, suggesting that SnRK2-mediated phosphorylation may regulate this interaction. These results indicate that phosphorylation domains of OREB1 are not functionally redundant and regulate at least three different functions, including transactivation activity, DNA binding, and protein interactions. The multisite phosphorylation of OREB1 is likely a key for the fine control of its activity and signal integration in the complex stress signaling network of plant cells.