Glutathione-S-transferase P1 is a critical regulator of Cdk5 kinase activity

Cyclin dependent kinase-5 (Cdk5) activity is deregulated in Alzheimer's disease (AD) and contributes to all three hallmarks: neurotoxic β-amyloid formation, neurofibrillary tangles, and neuronal death. However, the mechanism leading to Cdk5 deregulation remains controversial. Cdk5 deregulation in AD is usually linked to the formation of p25, a proteolysis product of Cdk5 activator p35, which leads to Cdk5 mislocalization and hyperactivation. A few studies have indeed shown increased p25 levels in AD brains; however, others have refuted this observation. These contradictory findings suggest that additional factors contribute to Cdk5 deregulation. This study identified glutathione-S-transferase pi 1 (GSTP1) as a novel Cdk5 regulatory protein. We demonstrate that it is a critical determinant of Cdk5 activity in human AD brains and various cancer and neuronal cells. Increased GSTP1 levels were consistently associated with reduced Cdk5 activity. GSTP1 directly inhibits Cdk5 by dislodging p25/p35, and indirectly by eliminating oxidative stress. Cdk5 promotes and is activated by oxidative stress, thereby engaging a feedback loop which ultimately leads to cell death. Not surprisingly, GSTP1 transduction conferred a high degree of neuroprotection under neurotoxic conditions. Given the critical role of oxidative stress in AD pathogenesis, an increase in GSTP1 level may be an alternative way to modulate Cdk5 signaling, eliminate oxidative stress, and prevent neurodegeneration.     

Gfer is a critical regulator of HSC proliferation

Hematopoietic stem cells (HSC) are a relatively quiescent pool of cells that perform the arduous task of replacing the short-lived mature cells of the peripheral blood. While a rapid expansion of HSCs under periods of hematological stress is warranted, their enhanced proliferation during homeostasis leads to loss of function. We recently reported that in HSCs, the evolutionarily conserved growth factor erv1-like (Gfer) acts to counter jun activation domain-binding protein 1 (Jab1)-mediated nuclear export and destabilization of the cell cycle inhibitor, p27^kip1, by directly binding to and sequestering the COP9 signalosome (CSN) subunit. Through this mechanism, Gfer promotes quiescence and maintains the functional integrity of HSCs. Here, we extend our study to demonstrate an association between Gfer and Ca²⁺/calmodulin-dependent protein kinase IV (CaMKIV) in the regulation of HSC proliferation. Highly proliferative and functionally deficient Camk4^-/- HSCs possess significantly lower levels of Gfer and p27^kip1. Ectopic expression of Gfer restores quiescence and elevates p27^kip1 expression in Camk4^-/- HSCs. These results further substantiate a critical role for Gfer in the restriction of unwarranted proliferation in HSCs through the inhibition of Jab1 and subsequent stabilization and nuclear retention of p27^kip1. This Gfer-mediated pro-quiescence mechanism could be therapeutically exploited in the treatment of hematological malignancies associated with elevated Jab1 and reduced p27^kip1.Key words: hematopoietic stem cells, quiescence, proliferation, Gfer, CaMKIV, Jab1, p27^kip1, Bcl-2     

Gfer is a critical regulator of HSC proliferation

Hematopoietic stem cells (HSC) are a relatively quiescent pool of cells that perform the arduous task of replacing the short-lived mature cells of the peripheral blood. While a rapid expansion of HSCs under periods of hematological stress is warranted, their enhanced proliferation during homeostasis leads to loss of function. We recently reported that in HSCs, the evolutionarily conserved growth factor erv1-like (Gfer) acts to counter jun activation domain-binding protein 1 (Jab1)-mediated nuclear export and destabilization of the cell cycle inhibitor, p27^kip1, by directly binding to and sequestering the COP9 signalosome (CSN) subunit. Through this mechanism, Gfer promotes quiescence and maintains the functional integrity of HSCs. Here, we extend our study to demonstrate an association between Gfer and Ca²⁺/calmodulin-dependent protein kinase IV (CaMKIV) in the regulation of HSC proliferation. Highly proliferative and functionally deficient Camk4^-/- HSCs possess significantly lower levels of Gfer and p27^kip1. Ectopic expression of Gfer restores quiescence and elevates p27^kip1 expression in Camk4^-/- HSCs. These results further substantiate a critical role for Gfer in the restriction of unwarranted proliferation in HSCs through the inhibition of Jab1 and subsequent stabilization and nuclear retention of p27^kip1. This Gfer-mediated pro-quiescence mechanism could be therapeutically exploited in the treatment of hematological malignancies associated with elevated Jab1 and reduced p27^kip1.     

DAPK2 is a novel regulator of mTORC1 activity and autophagy

Autophagy is a tightly regulated catabolic process, which is upregulated in cells in response to many different stress signals. Inhibition of mammalian target of rapmaycin complex 1 (mTORC1) is a crucial step in induction of autophagy, yet the mechanisms regulating the fine tuning of its activity are not fully understood. Here we show that death-associated protein kinase 2 (DAPK2), a Ca²⁺-regulated serine/threonine kinase, directly interacts with and phosphorylates mTORC1, and has a part in suppressing mTOR activity to promote autophagy induction. DAPK2 knockdown reduced autophagy triggered either by amino acid deprivation or by increases in intracellular Ca²⁺ levels. At the molecular level, DAPK2 depletion interfered with mTORC1 inhibition caused by these two stresses, as reflected by the phosphorylation status of mTORC1 substrates, ULK1 (unc-51-like kinase 1), p70 ribosomal S6 kinase and eukaryotic initiation factor 4E-binding protein 1. An increase in mTORC1 kinase activity was also apparent in unstressed cells that were depleted of DAPK2. Immunoprecipitated mTORC1 from DAPK2-depleted cells showed increased kinase activity in vitro, an indication that DAPK2 regulation of mTORC1 is inherent to the complex itself. Indeed, we found that DAPK2 associates with components of mTORC1, as demonstrated by co-immunoprecipitation with mTOR and its complex partners, raptor (regulatory-associated protein of mTOR) and ULK1. DAPK2 was also able to interact directly with raptor, as shown by recombinant protein-binding assay. Finally, DAPK2 was shown to phosphorylate raptor in vitro. This phosphorylation was mapped to Ser721, a site located within a highly phosphorylated region of raptor that has previously been shown to regulate mTORC1 activity. Thus, DAPK2 is a novel kinase of mTORC1 and is a potential new member of this multiprotein complex, modulating mTORC1 activity and autophagy levels under stress and steady-state conditions.Macroautophagy (hereafter referred to as autophagy) is a highly regulated intracellular bulk degradation process found ubiquitously in eukaryotes. During autophagy a double-membrane vesicle, termed an autophagosome, engulfs cytoplasmic materials, including whole organelles. The autophagosome is later fused with the lysosome and its content degraded by hydrolases.¹ Basal levels of autophagy are maintained within the cell during steady state, and are involved in cell homeostasis activities such as turnover of long-lived proteins, preventing accumulation of protein aggregates, and removal of damaged cellular structures.² Beyond this homeostatic function, autophagy is stimulated during various stress conditions, such as nutrient deprivation, intracellular Ca²⁺ increase, hypoxia, ER stress and oxidative stress, to ensure continuous cell survival under stress.³A critical step in the induction of autophagy comprises the inactivation of a key negative regulator of the process, the mammalian target of rapamycin (mTOR).⁴ mTOR is a conserved serine/threonine protein kinase that acts as a master regulator in the cell. mTOR forms a rapamycin-sensitive complex named mTORC1 with its binding partner raptor (regulatory-associated protein of mTOR), which mediates mTOR''s substrate presentation.⁵ mTORC1 senses nutrient availability, growth factors and energy levels, and, in response, regulates cell growth, metabolism and protein synthesis, mainly by phosphorylation of substrates involved in protein translation: the p70 ribosomal S6 kinase (p70S6K) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). Under nutrient-rich conditions, mTORC1 suppresses autophagy to basal levels by phosphorylating and inhibiting the autophagy proteins ULK1 (unc-51-like kinase 1) and Atg13. Upon autophagic stimulus, mTORC1 activity is inhibited and the ULK1 complex is activated, leading to autophagy induction.⁶ The activity levels of mTORC1 are regulated by several mechanisms, such as interacting proteins, cellular localization and phosphorylation events. Raptor phosphorylation has been suggested as a mechanism by which upstream kinases such as AMPK,⁷ RSK⁸ and ULK1⁹ can regulate mTORC1 activity.Death-associated protein kinase 2 (DAPK2; also named DRP-1) is a 42-kDa Ca²⁺/calmodulin (CaM)-regulated serine/threonine kinase,¹⁰ and a closely related homolog of DAPK, a gene originally discovered in an attempt to find positive regulators of cell death.¹¹ DAPK2 was identified based on homology to the catalytic domain of DAPK. DAPK2 is a soluble cytoplasmatic protein, which triggers massive membrane blebbing and appearance of double-membrane autophagic vesicles upon its overexpression (for a review see Shiloh et al.¹²). DAPK2''s substrates and interacting proteins are mostly unknown, with the exception of the myosin II regulatory light chain, which has been shown to be an in vitro and in vivo substrate.¹³ Although many publications have studied DAPK, its substrates and its role in cell death and autophagy,^{14, 15} very little is known about DAPK2 substrates, cellular functions or the molecular pathways that it regulates.In this work, we studied the involvement of DAPK2 in the autophagic module. We identified DAPK2 as a novel interacting protein of mTORC1, and as a negative regulator of the complex both during steady-state growth conditions and in response to different stress autophagic signals. We identified mTOR''s binding partner, raptor, as a substrate of DAPK2, and found Ser721 as its phosphorylation site.     

A novel allele in the promoter of the hepatic lipase is associated with increased concentration of HDL-C and decreased promoter activity

Hepatic lipase (HL) is a lipolytic enzyme involved in the metabolism of plasma lipoproteins, especially HDLs. Association of the polymorphisms in the promoter region of the LIPC gene to post-heparin plasma HL activity and the plasma HDL-C concentration has been investigated thoroughly, but to date little is known about this in the Chinese. In the present study, we analyzed the polymorphisms in the promoter region of LIPC gene in Chinese patients with coronary artery disease (CAD) using denaturing high performance liquid chromatography (DHPLC) and DNA sequencing. As the result, a novel single nucleotide polymorphism -586T-to-C was identified and no linkage of this variant with other polymorphisms in the promoter was found. Compared with the nonsymptomatic control subjects, excess of carriers of the -586T/C substitution were detected in the CAD patients (43% vs. 31%, chi(2) = 4.597, degree of freedom = 2, P = 0.032).The -586C allele carriers in the CAD patients had a significantly higher HDL-C level than the noncarriers (1.13 +/- 0.24 mmol/l vs. 0.91 +/- 0.14 mmol/l, P < 0.05). To test the functionality of this substitution, luciferase-reporter assays was performed in HepG2 cells. Promoter activity of the -586C construct was decreased 2-fold than the -586T construct. Our studies suggest that a T-to-C substitution at -586 of the LIPC promoter is associated with a lowered HL activity and that this variation may contribute to the increased plasma HDL-C concentration in the Chinese.     

GEMC1 is a critical regulator of multiciliated cell differentiation

The generation of multiciliated cells (MCCs) is required for the proper function of many tissues, including the respiratory tract, brain, and germline. Defects in MCC development have been demonstrated to cause a subclass of mucociliary clearance disorders termed reduced generation of multiple motile cilia (RGMC). To date, only two genes, Multicilin (MCIDAS) and cyclin O (CCNO) have been identified in this disorder in humans. Here, we describe mice lacking GEMC1 (GMNC), a protein with a similar domain organization as Multicilin that has been implicated in DNA replication control. We have found that GEMC1‐deficient mice are growth impaired, develop hydrocephaly with a high penetrance, and are infertile, due to defects in the formation of MCCs in the brain, respiratory tract, and germline. Our data demonstrate that GEMC1 is a critical regulator of MCC differentiation and a candidate gene for human RGMC or related disorders.     

A novel alpha-helix in the first fibronectin type III repeat of the neural cell adhesion molecule is critical for N-glycan polysialylation

Polysialic acid is a developmentally regulated, anti-adhesive glycan that is added to the neural cell adhesion molecule, NCAM. Polysialylated NCAM is critical for brain development and plays roles in synaptic plasticity, axon guidance, and cell migration. The first fibronectin type III repeat of NCAM, FN1, is necessary for the polysialylation of N-glycans on the adjacent immunoglobulin domain. This repeat cannot be replaced by other fibronectin type III repeats. We solved the crystal structure of human NCAM FN1 and found that, in addition to a unique acidic surface patch, it possesses a novel alpha-helix that links strands 4 and 5 of its beta-sandwich structure. Replacement of the alpha-helix did not eliminate polysialyltransferase recognition, but shifted the addition of polysialic acid from the N-glycans modifying the adjacent immunoglobulin domain to O-glycans modifying FN1. Other experiments demonstrated that replacement of residues in the acidic surface patch alter the polysialylation of both N- and O-glycans in the same way, while the alpha-helix is only required for the polysialylation of N-glycans. Our data are consistent with a model in which the FN1 alpha-helix is involved in an Ig5-FN1 interaction that is critical for the correct positioning of Ig5 N-glycans for polysialylation.     

A conserved DYW domain of the pentatricopeptide repeat protein possesses a novel endoribonuclease activity

Many plant pentatricopeptide repeat (PPR) proteins are known to contain a highly conserved C-terminal DYW domain whose function is unknown. Recently, the DYW domain has been proposed to play a role in RNA editing in plant organelles. To address this possibility, we prepared recombinant DYW proteins and tested their cytidine deaminase activity. However, we could not detect any activity in the assays we used. Instead, we found that the recombinant DYW domains possessed endoribonuclease activity and cleaved before adenosine residues in the RNA molecule. Some DYW-containing PPR proteins may catalyze site-specific cleavage of target RNA species.     

Spatiotemporally regulated protein kinase A activity is a critical regulator of growth factor-stimulated extracellular signal-regulated kinase signaling in PC12 cells

PC12 cells exhibit precise temporal control of growth factor signaling in which stimulation with epidermal growth factor (EGF) leads to transient extracellular signal-regulated kinase (ERK) activity and cell proliferation, whereas nerve growth factor (NGF) stimulation leads to sustained ERK activity and differentiation. While cyclic AMP (cAMP)-mediated signaling has been shown to be important in conferring the sustained ERK activity achieved by NGF, little is known about the regulation of cAMP and cAMP-dependent protein kinase (PKA) in these cells. Using fluorescence resonance energy transfer (FRET)-based biosensors localized to discrete subcellular locations, we showed that both NGF and EGF potently activate PKA at the plasma membrane, although they generate temporally distinct activity patterns. We further show that both stimuli fail to induce cytosolic PKA activity and identify phosphodiesterase 3 (PDE3) as a critical regulator in maintaining this spatial compartmentalization. Importantly, inhibition of PDE3, and thus perturbation of the spatiotemporal regulation of PKA activity, dramatically increases the duration of EGF-stimulated nuclear ERK activity in a PKA-dependent manner. Together, these findings identify EGF and NGF as potent activators of PKA activity specifically at the plasma membrane and reveal a novel regulatory mechanism contributing to the growth factor signaling specificity achieved by NGF and EGF in PC12 cells.     

Drosophila Ndfip is a novel regulator of Notch signaling

In the Drosophila wing, the Nedd4 ubiquitin ligases (E3s), dNedd4 and Su(dx), are important negative regulators of Notch signaling; they ubiquitinate Notch, promoting its endocytosis and turnover. Here, we show that Drosophila Nedd4 family interacting protein (dNdfip) interacts with the Drosophila Nedd4-like E3s. dNdfip expression dramatically enhances dNedd4 and Su(dx)-mediated wing phenotypes and further disrupts Notch signaling. dNdfip colocalizes with Notch in wing imaginal discs and with the late endosomal marker Rab7 in cultured cells. In addition, dNdfip expression in the wing leads to ectopic Notch signaling. Supporting this, expression of dNdfip suppressed Notch(+/-) wing phenotype and knockdown of dNdfip enhanced the Notch(+/-) wing phenotype. The increase in Notch activity by dNdfip is ligand independent as dNdfip expression also suppressed deltex RNAi and Serrate(+/-) wing phenotypes. The opposing effects of dNdfip expression on Notch signaling and its late endosomal localization support a model whereby dNdfip promotes localization of Notch to the limiting membrane of late endosomes allowing for activation, similar to the model previously shown with ectopic Deltex expression. When dNedd4 or Su(dx) are also present, dNdfip promotes their activity in Notch ubiquitination and internalization to the lysosomal lumen for degradation.     

alpha -melanocyte-stimulating hormone is a novel regulator of bone

alpha-Melanocyte-stimulating hormone (alpha-MSH), a 13-amino acid peptide produced in the brain and pituitary gland, is a regulator of appetite and body weight, and its production is regulated by leptin, a factor that affects bone mass when administered centrally. alpha-MSH acts via melanocortin receptors. Humans deficient in melanocortin receptor 4 (MC4-R) have increased bone mass, and MC4-R has been identified in an osteoblast-like cell line. Thus alpha-MSH may act directly on the skeleton, a question addressed by the present studies. In primary cultures of osteoblasts and chondrocytes, alpha-MSH dose dependently (>or=10(-9) M) stimulated cell proliferation. In bone marrow cultures, alpha-MSH (>10(-9) M) stimulated osteoclastogenesis. Systemic administration of alpha-MSH to mice (20 injections of 4.5 microg/day) decreased the trabecular bone volume in the proximal tibiae from 19.5 +/- 1.8 to 15.2 +/- 1.4% (P = 0.03) and reduced trabecular number (P = 0.001). Radiographic indexes of trabecular bone, assessed by phase-contrast X-ray imaging, confirmed the bone loss. It is concluded that alpha-MSH acts directly on bone, increasing bone turnover, and, when administered systemically, it decreases bone volume. The latter result may also be contributed to by alpha-MSH effects elsewhere, such as the adipocyte, pancreatic beta-cell, or central nervous system.