Pin1-dependent prolyl isomerization modulates the stress-induced phosphorylation of high molecular weight neurofilament protein

Aberrant phosphorylation of neuronal cytoskeletal proteins is a key pathological event in neurodegenerative disorders such as Alzheimer disease (AD) and amyotrophic lateral sclerosis, but the underlying mechanisms are still unclear. Previous studies have shown that Pin1, a peptidylprolyl cis/trans-isomerase, may be actively involved in the regulation of Tau hyperphosphorylation in AD. Here, we show that Pin1 modulates oxidative stress-induced NF-H phosphorylation. In an in vitro kinase assay, the addition of Pin1 substantially increased phosphorylation of NF-H KSP repeats by proline-directed kinases, Erk1/2, Cdk5/p35, and JNK3 in a concentration-dependent manner. In vivo, dominant-negative (DN) Pin1 and Pin1 small interfering RNA inhibited epidermal growth factor-induced NF-H phosphorylation. Because oxidative stress plays an important role in the pathogenesis of neurodegenerative diseases, we studied the role of Pin1 in stressed cortical neurons and HEK293 cells. Both hydrogen peroxide (H(2)O(2)) and heat stresses induce phosphorylation of NF-H in transfected HEK293 cells and primary cortical cultures. Knockdown of Pin1 by transfected Pin1 short interference RNA and DN-Pin1 rescues the effect of stress-induced NF-H phosphorylation. The H(2)O(2) and heat shock induced perikaryal phospho-NF-H accumulations, and neuronal apoptosis was rescued by inhibition of Pin1 in cortical neurons. JNK3, a brain-specific JNK isoform, is activated under oxidative and heat stresses, and inhibition of Pin1 by Pin1 short interference RNA and DN-Pin1 inhibits this pathway. These results implicate Pin1 as a possible modulator of stress-induced NF-H phosphorylation as seen in neurodegenerative disorders like AD and amyotrophic lateral sclerosis. Thus, Pin1 may be a potential therapeutic target for these diseases.     

Proline-directed phosphorylation and isomerization in mitotic regulation and in Alzheimer's Disease

The reversible phosphorylation of proteins on serine/threonine residues preceding proline (Ser/Thr-Pro) is a major regulatory mechanism for the control of a series of cell cycle events. Although phosphorylation is thought to regulate protein function by inducing conformational changes, little is known about most of these conformational changes and their significance. Recent studies indicate that the conformation and function of a subset of these phosphorylated proteins are controlled by the prolyl isomerase Pin1 through isomerization of specific phosphorylated Ser/Thr-Pro bonds. Furthermore, compelling evidence supports the idea that proline-directed phosphorylation and subsequent isomerization play a critical role not only in cell cycle control, but also in the development of Alzheimer's disease, where postmitotic neurons display various cell cycle markers, especially mitotic events, prior to degeneration.     

Phosphorylation of the head domain of neurofilament protein (NF-M): a factor regulating topographic phosphorylation of NF-M tail domain KSP sites in neurons

In neurons the phosphorylation of neurofilament (NF) proteins NF-M and NF-H is topographically regulated. Although kinases and NF subunits are synthesized in cell bodies, extensive phosphorylation of the KSP repeats in tail domains of NF-M and NF-H occurs primarily in axons. The nature of this regulation, however, is not understood. As obligate heteropolymers, NF assembly requires interactions between the core NF-L with NF-M or NF-H subunits, a process inhibited by NF head domain phosphorylation. Phosphorylation of head domains at protein kinase A (PKA)-specific sites seems to occur transiently in cell bodies after NF subunit synthesis. We have proposed that transient phosphorylation of head domains prevents NF assembly in the soma and inhibits tail domain phosphorylation; i.e. assembly and KSP phosphorylation in axons depends on prior dephosphorylation of head domain sites. Deregulation of this process leads to pathological accumulations of phosphorylated NFs in the soma as seen in some neurodegenerative disorders. To test this hypothesis, we studied the effect of PKA phosphorylation of the NF-M head domain on phosphorylation of tail domain KSP sites. In rat cortical neurons we showed that head domain phosphorylation of endogenous NF-M by forskolin-activated PKA inhibits NF-M tail domain phosphorylation. To demonstrate the site specificity of PKA phosphorylation and its effect on tail domain phosphorylation, we transfected NIH3T3 cells with NF-M mutated at PKA-specific head domain serine residues. Epidermal growth factor stimulation of cells with mutant NF-M in the presence of forskolin exhibited no inhibition of NF-tail domain phosphorylation compared with the wild type NF-M-transfected cells. This is consistent with our hypothesis that transient phosphorylation of NF-M head domains inhibits tail domain phosphorylation and suggests this as one of several mechanisms underlying topographic regulation.     

Myelin-associated glycoprotein modulates expression and phosphorylation of neuronal cytoskeletal elements and their associated kinases

Decreased phosphorylation of neurofilaments in mice lacking myelin-associated glycoprotein (MAG) was shown to be associated with decreased activities of extracellular-signal regulated kinases (ERK1/2) and cyclin-dependent kinase-5 (cdk5). These in vivo changes could be caused directly by the absence of a MAG-mediated signaling pathway or secondary to a general disruption of the Schwann cell-axon junction that prevents signaling by other molecules. Therefore, in vitro experimental paradigms of MAG interaction with neurons were used to determine if MAG directly influences expression and phosphorylation of cytoskeletal proteins and their associated kinases. COS-7 cells stably transfected with MAG or with empty vector were co-cultured with primary dorsal root ganglion (DRG) neurons. Total amounts of the middle molecular weight neurofilament subunit (NF-M), microtubule-associated protein 1B (MAP1B), MAP2, and tau were up-regulated significantly in DRG neurons in the presence of MAG. There was also increased expression of phosphorylated high molecular weight neurofilament subunit (NF-H), NF-M, and MAP1B. Additionally, in similar in vitro paradigms, total and phosphorylated NF-M were increased significantly in PC12 neurons co-cultured with MAG-expressing COS cells or treated with a soluble MAG Fc-chimera. The increased expression of phosphorylated cytoskeletal proteins in the presence of MAG in vitro was associated with increased activities of ERK 1/2 and cdk5. We propose that interaction of MAG with an axonal receptor(s) induces a signal transduction cascade that regulates expression of cytoskeletal proteins and their phosphorylation by these proline-directed protein kinases.     

Pathways to motor neuron degeneration in transgenic mouse models

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurological disorder characterized by the selective loss of motor neurons. A pathological hallmark of both sporadic and familial ALS is the presence of abnormal accumulations of neurofilament and peripherin proteins in motor neurons. In the past decade, transgenic mouse approaches have been used to address the role of such cytoskeletal abnormalities in motor neuron disease and also to unravel the pathogenesis caused by mutations in the gene coding for superoxide dismutase 1 (SOD1) that account for ~20% of familial ALS cases. In mouse models, disparate effects could result from different types of intermediate filament (IF) aggregates. Perikaryal IF accumulations induced by the overexpression of any of the three wild-type neurofilament proteins were quite well tolerated by motor neurons. Indeed, perikaryal swellings provoked by NF-H overexpression can even confer protection against toxicity of mutant SOD1. Other types of IF aggregates seem neurotoxic, such as those found in transgenic mice overexpressing either peripherin or an assembly-disrupting NF-L mutant. Moreover, understanding the toxicity of SOD1 mutations has been surprisingly difficult. The analysis of transgenic mice expressing mutant SOD1 has yielded complex results, suggesting that multiple pathways may contribute to disease that include the involvement of non-neuronal cells.     

Phosphorylation-specific prolyl isomerization: is there an underlying theme?

The prolyl isomerase Pin1 is a conserved enzyme that is intimately involved in diverse biological processes and pathological conditions such as cancer and Alzheimer's disease. By catalysing cis-trans interconversion of certain motifs containing phosphorylated serine or threonine residues followed by a proline residue (pSer/Thr-Pro), Pin1 can have profound effects on phosphorylation signalling. The structural and functional differences that result from cis-trans isomerization of specific pSer/Thr-Pro motifs probably underlie most, if not all, Pin1-dependent actions. Phosphorylation-dependent prolyl isomerization by Pin1 remains a unique mode for the modulation of signal transduction. Here, we provide an overview of the plethora of regulatory events that involve this unique enzyme, with a particular focus on oncogenic signalling and neurodegeneration.     

Phosphorylation of AATYK1 by Cdk5 Suppresses Its Tyrosine Phosphorylation

Apoptosis-associated tyrosine kinase 1 (AATYK1), a novel serine/threonine kinase that is highly expressed in the brain, is involved in neurite extension and apoptosis of cerebellar granule neurons; however, its precise function remains unknown. In this study, we investigated the interaction of AATYK1A with Cyclin-dependent kinase 5 (Cdk5)/p35, a proline-directed protein kinase that is predominantly expressed in neurons. AATYK1A bound to the p35 activation subunit of Cdk5 in cultured cells and in mouse brains and colocalized with p35 on endosomes in COS-7 cells. AATYK1A was phosphorylated at Ser34 by Cdk5/p35 in vitro, in cultured neurons and in mouse brain. In PC12D cells, Ser34 phosphorylation increased after treatment with nerve growth factor and phosphorylated AATYK1A accumulated in growth cones of PC12D cells. Ser34 phosphorylation suppressed the tyrosine phosphorylation of AATYK1A by Src family kinases. These results suggest a possibility that AATYK1A plays a role in early to recycling endosomes and its function is regulated by phosphorylation with Cdk5 or Src-family kinases.     

Pin1 Promotes Transforming Growth Factor-��-induced Migration and Invasion

Transforming growth factor-β (TGF-β) regulates a wide variety of biological activities. It induces potent growth-inhibitory responses in normal cells but promotes migration and invasion of cancer cells. Smads mediate the TGF-β responses. TGF-β binding to the cell surface receptors leads to the phosphorylation of Smad2/3 in their C terminus as well as in the proline-rich linker region. The serine/threonine phosphorylation sites in the linker region are followed by the proline residue. Pin1, a peptidyl-prolyl cis/trans isomerase, recognizes phosphorylated serine/threonine-proline motifs. Here we show that Smad2/3 interacts with Pin1 in a TGF-β-dependent manner. We further show that the phosphorylated threonine 179-proline motif in the Smad3 linker region is the major binding site for Pin1. Although epidermal growth factor also induces phosphorylation of threonine 179 and other residues in the Smad3 linker region the same as TGF-β, Pin1 is unable to bind to the epidermal growth factor-stimulated Smad3. Further analysis suggests that phosphorylation of Smad3 in the C terminus is necessary for the interaction with Pin1. Depletion of Pin1 by small hairpin RNA does not significantly affect TGF-β-induced growth-inhibitory responses and a number of TGF-β/Smad target genes analyzed. In contrast, knockdown of Pin1 in human PC3 prostate cancer cells strongly inhibited TGF-β-mediated migration and invasion. Accordingly, TGF-β induction of N-cadherin, which plays an important role in migration and invasion, is markedly reduced when Pin1 is depleted in PC3 cells. Because Pin1 is overexpressed in many cancers, our findings highlight the importance of Pin1 in TGF-β-induced migration and invasion of cancer cells.     

Topographic regulation of neuronal intermediate filaments by phosphorylation, role of peptidyl-prolyl isomerase 1: significance in neurodegeneration

The neuronal cytoskeleton is tightly regulated by phosphorylation and dephosphorylation reactions mediated by numerous associated kinases, phosphatases and their regulators. Defects in the relative kinase and phosphatase activities and/or deregulation of compartment-specific phosphorylation result in neurodegenerative disorders. The largest family of cytoskeletal proteins in mammalian cells is the superfamily of intermediate filaments (IFs). The neurofilament (NF) proteins are the major IFs. Aggregated forms of hyperphosphorylated tau and phosphorylated NFs are found in pathological cell body accumulations in the central nervous system of patients suffering from Alzheimer’s disease, Parkinson’s disease, and Amyotrophic Lateral Sclerosis. The precise mechanisms for this compartment-specific phosphorylation of cytoskeletal proteins are not completely understood. In this review, we focus on the mechanisms of neurofilament phosphorylation in normal physiology and neurodegenerative diseases. We also address the recent breakthroughs in our understanding the role of different kinases and phosphatases involved in regulating the phosphorylation status of the NFs. In addition, special emphasis has been given to describe the role of phosphatases and Pin1 in phosphorylation of NFs.     

Phosphorylation and dephosphorylation of distinct isoforms of the heavy neurofilament protein NF-H

Summary 1. Previous immunohistochemical studies led to the suggestion that distinctly phosphorylated neurofilament isoforms exist in different types of neurons. We have recently examined this hypothesis by direct biochemical experiments, which revealed that the heavy neurofilament protein NF-H of bovine ventral root cholinergic neurons is more acidic and markedly more phosphorylated than that of bovine dorsal root neurons.2. In the present study we employed this system to study the degree to which distinctly phosphorylated NF-H isoforms differ in the extents to which they can be phosphorylated and dephosphorylatedin vitro. This was performed utilizing alkaline phosphatase and protein kinase PK40^ERK, which is specific to serines of Lys-Ser-Pro (KSP) repeats. The results obtained reveal that:3. The more extensively phosphorylated ventral root NF-H is dephosphorylated more rapidly than dorsal root NF-H.4. Ventral root NF-H and dorsal root NF-H in their native form are both poor substrates of PK40^ERK.5. Following dephosphorylation, ventral root and dorsal root NF-H are phosphorylated extensively and differentially by this kinase. Under these conditions, PK40^ERK catalyzes the incorporation of, respectively, 4.2±1.3 and 2.8±0.6 mol of phosphate per molecule of ventral root NF-H and dorsal root NF-H. The ratio of phosphates incorporated into ventral root NF-H to those incorporated into dorsal root NF-H is 1.46±0.17.6. These findings support the hypothesis that different classes of neurons contain distinctly phosphorylated neurofilaments and show that ventral root and dorsal root neurons are a useful model system for studying the distinct characteristics of neurofilament phosphorylation in different types of neurons.     

Pinning down cell signaling, cancer and Alzheimer's disease

Protein phosphorylation on certain serine or threonine residues preceding proline (Ser/Thr-Pro) is a pivitol signaling mechanism in diverse cellular processes and its deregulation can lead to human disease. However, little is known about how these phosphorylation events actually control cell signaling. Pin1 is a highly conserved enzyme that isomerizes only the phosphorylated Ser/Thr-Pro bonds in certain proteins, thereby inducing conformational changes. Recent results indicate that such conformational changes following phosphorylation are a novel signaling mechanism pivotal in regulating many cellular functions. This mechanism also offers new insights into the pathogenesis and treatment of human disease, most notably cancer and Alzheimer's disease. Thus, Pin1 plays a key role in linking signal transduction to the pathogenesis of cancer and Alzheimer's disease - two major age-related diseases.     

Pinning down phosphorylated tau and tauopathies

Neurofibrillary tangles (NFTs) are prominent neuronal lesions in a large subset of neurodegenerative diseases, including Alzheimer's disease (AD). NFTs are mainly composed of insoluble Tau that is hyperphosphorylated on many serine or threonine residues preceding proline (pSer/Thr-Pro). Tau hyperphosphorylation abolishes its biological function to bind microtubules and promotes microtubule assembly and precedes neurodegeneration. Not much is known about how tau is further regulated following phosphorylation. Notably, we have recently shown that phosphorylated Ser/Thr-Pro motifs exist in two distinct conformations. The conversion between two conformations in some proteins is catalyzed by the prolyl isomerase Pin1. Pin1 binds to tau phosphorylated specifically on the Thr231-Pro site and probably catalyzes cis/trans isomerization of pSer/Thr-Pro motif(s), thereby inducing conformational changes in tau. Such conformational changes can directly restore the ability of phosphorylated Tau to bind microtubules and promote microtubule assembly and/or facilitate tau dephosphorylation by its phosphatase PP2A, as PP2A activity is conformation-specific. Furthermore, Pin1 expression inversely correlates with the predicted neuronal vulnerability in normally aged brain and also with actual neurofibrillary degeneration in AD brain. Moreover, deletion of the gene encoding Pin1 in mice causes progressive age-dependent neuropathy characterized by motor and behavioral deficits, tau hyperphosphorylation, tau filament formation and neuronal degeneration. Distinct from all other mouse models where transgenic overexpression of specific proteins elicits tau-related pathologies, Pin1 is the first protein whose depletion causes age-dependent neurodegeneration and tau pathologies. Thus, Pin1 is pivotal in maintaining normal neuronal function and preventing age-dependent neurodegeneration. This could represent a promising interventive target to prevent neurodegenerative diseases.     

Identification of Endogenously Phosphorylated KSP Sites in the High-Molecular-Weight Rat Neurofilament Protein

Abstract: The high-molecular-weight neurofilament protein (NF-H) is highly phosphorylated in vivo, with estimates as high as 16–51 mol of P_i/mol of protein. Most of the phosphorylation sites are thought to be located on Ser residues in multiple KSP repeats, in the carboxy-terminal tail region of the molecule. Because the extent and site-specific patterns of tail domain phosphorylation are believed to modulate neurofilament structure and function, it becomes essential to identify the endogenous sites of phosphorylation. In this study, we have used selective proteolytic cleavage procedures, P_i determinations, microsequencing, and mass-spectral analysis to determine the endogenously phosphorylated sites in the NF-H tail isolated from rat spinal cord. Twenty Ser residues in NF-H carboxy-terminal tail were analyzed; nine of these, all located in KSP repeats, were phosphorylated. No detectable phosphorylation could be identified in any of the 11 "non-KSP" Ser residues that were examined. KSPXKX, KSPXXX, and KSPXXK motifs were found to be phosphorylated. In addition, a 27-kDa KSP-rich domain, containing 43 virtually uninterrupted KSPXXX repeats, was isolated from the tail domain and found to contain between 30 and 35 mol of P_i/mol of protein. This domain appeared to be highly resistant to endoproteinase Glu-C digestion, although it contains a large number of glutamate residues. It could be proteolyzed, however, after dephosphorylation. This suggests that phosphorylation of the tail domain may contribute to neurofilament stability in vivo. A neuronal-derived protein kinase that specifically phosphorylates only KSPXKX motifs in neurofilaments has been reported. The presence of extensively phosphorylated KSPXXX repeats in NF-H in vivo suggests the existence of yet another, unidentified kinase(s) with specificity for KSPXXX motifs.     

Pinning down proline-directed phosphorylation signaling

The reversible phosphorylation of proteins on serine or threonine residues preceding proline (Ser/Thr-Pro) is a major cellular signaling mechanism. Although it is proposed that phosphorylation regulates the function of proteins by inducing a conformational change, there are few clues about the actual conformational changes and their importance. Recent identification of the novel prolyl isomerase Pin1 that specifically isomerizes only the phosphorylated Ser/Thr-Pro bonds in certain proteins led us to propose a new signaling mechanism, whereby prolyl isomerization catalytically induces conformational changes in proteins following phosphorylation to regulate protein function. Emerging data indicate that such conformational changes have profound effects on catalytic activity, dephosphorylation, protein-protein interactions, subcellular location and/or turnover. Furthermore, this post-phosphorylation mechanism might play an important role in cell growth control and diseases such as cancer and Alzheimer's.     

Neurofilament subunit NF-H modulates axonal diameter by selectively slowing neurofilament transport

To examine the mechanism through which neurofilaments regulate the caliber of myelinated axons and to test how aberrant accumulations of neurofilaments cause motor neuron disease, mice have been constructed that express wild-type mouse NF-H up to 4.5 times the normal level. Small increases in NF-H expression lead to increased total neurofilament content and larger myelinated axons, whereas larger increases in NF-H decrease total neurofilament content and strongly inhibit radial growth. Increasing NF-H expression selectively slow neurofilament transport into and along axons, resulting in severe perikaryal accumulation of neurofilaments and proximal axonal swellings in motor neurons. Unlike the situation in transgenic mice expressing modest levels of human NF-H (Cote, F., J.F. Collard, and J.P. Julien. 1993. Cell. 73:35-46), even 4.5 times the normal level of wild-type mouse NF-H does not result in any overt phenotype or enhanced motor neuron degeneration or loss. Rather, motor neurons are extraordinarily tolerant of wild-type murine NF-H, whereas wild-type human NF-H, which differs from the mouse homolog at > 160 residue positions, mediates motor neuron disease in mice by acting as an aberrant, mutant subunit.     

Characterization of the phosphorylation sites of Mycobacterium tuberculosis serine/threonine protein kinases, PknA, PknD, PknE, and PknH by mass spectrometry

In Mycobacterium tuberculosis (Mtb), regulatory phosphorylation of proteins at serine and/or threonine residues by serine/threonine protein kinases (STPKs) is an emerging theme connected with the involvement of these enzymes in virulence mechanisms. The identification of phosphorylation sites in proteins provides a powerful tool to study signal transduction pathways and to identify the corresponding interaction networks. Detection of phosphorylated proteins as well as assignment of the phosphorylated sites in STPKs is a major challenge in proteomics since some of these enzymes might be interesting therapeutical targets. Using different strategies to identify phosphorylated residues, we report, in the present work, MS studies of the entire intracellular regions of recombinant protein kinases PknA, PknD, PknE, and PknH from Mtb. The on-target dephosphorylation/MALDI-TOF for identification of phosphorylated peptides was used in combination with LC-ESI/MS/MS for localization of phosphorylation sites. By doing so, seven and nine phosphorylated serine and/or threonine residues were identified as phosphorylation sites in the recombinant intracellular regions of PknA and PknH, respectively. The same technique led also to the identification of seven phosphorylation sites in each of the two recombinant kinases, PknD and PknE.