Parvathi Rudrabhatla

Cyclin-Dependent Kinase 5 Phosphorylation of Human Septin SEPT5 (hCDCrel-1) Modulates Exocytosis

Cyclin-dependent kinase 5 (Cdk5) is predominantly expressed in the nervous system, where it is involved in neuronal migration, synaptic transmission, and survival. The role of Cdk5 in synaptic transmission is mediated by regulating the cellular functions of presynaptic proteins such as synapsin, Munc18, and dynamin 1. Its multifunctional role at the synapse is complex and probably involves other novel substrates. To explore this possibility, we used a yeast two-hybrid screen of a human cDNA library with p35 as bait and isolated human septin 5 (SEPT5), known also as hCDCrel-1, as an interacting clone. Here we report that p35 associates with SEPT5 in GST (glutathione S-transferase)-pull-down and coimmunoprecipitation assays. We confirmed that Cdk5/p35 phosphorylates SEPT5 in vitro and in vivo and identified S327 of SEPT5 as a major phosphorylation site. A serine (S)-to-alanine (A) 327 mutant of SEPT5 bound syntaxin more efficiently than SEPT5 wild type. Additionally, coimmunoprecipitation from synaptic vesicle fractions and Cdk5 wild-type and knock-out lysates showed that phosphorylation of septin 5 by Cdk5/p35 decreases its binding to syntaxin-1. Moreover, mutant nonphosphorylated SEPT5 potentiated regulated exocytosis more than the wild type when each was expressed in PC12 cells. These data suggest that Cdk5 phosphorylation of human septin SEPT5 at S327 plays a role in modulating exocytotic secretion.

Quantitative phosphoproteomic analysis of neuronal intermediate filament proteins (NF-M/H) in Alzheimer's disease by iTRAQ

Aberrant hyperphosphorylation of neuronal cytoskeletal proteins is one of the major pathological hallmarks of neurodegenerative disorders such as Alzheimer disease (AD), amyotrophic lateral sclerosis (ALS), and Parkinsons disease (PD). Human NF‐M/H display a large number of multiple KSP repeats in the carboxy‐terminal tail domain, which are phosphorylation sites of proline‐directed serine/threonine (pSer/Thr‐Pro, KS/T‐P) kinases. The phosphorylation sites of NF‐M/H have not been characterized in AD brain. Here, we use quantitative phosphoproteomic methodology, isobaric tag for relative and absolute quantitation (iTRAQ), for the characterization of NF‐M/H phosphorylation sites in AD brain. We identified 13 hyperphosphorylated sites of NF‐M;9 Lys‐Ser‐Pro (KSP) sites;2 variant motifs, Glu‐Ser‐Pro (ESP) Ser‐736 and Leu‐Ser‐Pro (LSP) Ser‐837;and 2 non‐S/T‐P motifs, Ser‐783 and Ser‐788. All the Ser/Thr residues are phosphorylated at significantly greater abundance in AD brain compared with control brain. Ten hyperphosphorylated KSP sites have been identified on the C‐terminal tail domain of NF‐H, with greater abundance of phosphorylation in AD brain compared with control brain. Our data provide the direct evidence that NF‐M/H are hyperphosphorylated in AD compared with control brain and suggest the role of both proline‐directed and non‐proline‐directed protein kinases in AD. This study represents the first comprehensive iTRAQ analyses and quantification of phosphorylation sites of human NF‐M and NF‐H from AD brain and suggests that aberrant hyperphosphorylation of neuronal intermediate filament proteins is involved in AD.—Rudrabhatla, P., Grant, P., Jaffe, H., Strong, M. J., Pant, H. C. Quantitative phosphoproteomic analysis of neuronal intermediate filament proteins (NF‐M/H) in Alzheimers disease by iTRAQ. FASEB J. 24, 4396–4407 (2010). www.fasebj.org

Achaete-scute homologue-1 (ASH1) stimulates migration of lung cancer cells through Cdk5/p35 pathway

Cyclin-dependent kinase 5 (Cdk5) activity is important for the migration and invasion of cancer cells. Our results indicate that in the lung one of the mechanisms that hASH1 regulates—migration—takes place through induction of Cdk5 activity. Our data suggest that Cdk5 and its activator p35 promote lung cancer cell migration through hASH1-mediated signaling.

Phosphorylation-Specific Peptidyl-Prolyl Isomerization of Neuronal Cytoskeletal Proteins by Pin1: Implications for Therapeutics in Neurodegeneration

Pin1 [Protein Interacting with NIMA (never in mitosis A)] is a peptidyl prolyl cis-trans isomerase that isomerizes phospho-Serine/Threonine-Proline [p(S/T)-P] motifs of its target proteins. Pin1 functions in concert with proline directed kinases such as cyclin-dependent protein kinases, extracellular signal-regulated kinases, and c-Jun N- terminal kinase, and protein phosphatases such as protein phosphatase 2A (PP2A) and PP2B, in the regulation of a wide range of cellular processes including cell division, DNA damage response, and gene transcription, and in susceptibility to cancer and neurodegenerative diseases. This review focuses on the roles of Pin1 in neurodegenerative disorders including Alzheimers disease, Parkinsons disease, amyotrophic lateral sclerosis, and Frontotemporal dementia associated with parkinsonism linked to chromosome 17. Pin1 interacts with neuronal cytoskeletal proteins such as tau, amyloid-beta protein precursor, alpha-synuclein, and neurofilaments, often in association with phosphorylation events that influence their functions in the neuronal cytoskeleton. Overexpression of Pin1 reduces WT tau stability but increases P301L mutant tau stability. Pin1 associates with neurofilament H (NF-H) and modulates excitotoxic and oxidative stress induced perikaryal phosphorylation of NF-H. Pin1 mediates the neural specific apoptosis machinery. The specific inhibitors of Pin1 may have potential therapeutic implications in neurodegeneration.

Cyclin-dependent kinase 5 regulates E2F transcription factor through phosphorylation of Rb protein in neurons

Recent studies have shown the involvement of cyclin-dependent kinase 5 (Cdk5) in cell cycle regulation in postmitotic neurons. In this study, we demonstrate that Cdk5 and its co-activator p35 were detected in the nuclear fraction in neurons and Cdk5/p35 phosphorylated retinoblastoma (Rb) protein, a key protein controlling cell cycle re-entry. Cdk5/p35 phosphorylates Rb at the sites similar to those phosphorylated by Cdk4 and Cdk2. Furthermore, increased Cdk5 activity elevates activity of E2F transcription factor, which can trigger cell cycle re-entry, leading to neuronal cell death. A normal Cdk5 activity in neurons did not induce E2F activation, suggesting that Cdk5 does not induce cell cycle re-entry under normal conditions. Taken together, these results indicate that Cdk5 can regulate cell cycle by its ability to phosphorylate Rb. Most importantly, increased Cdk5 activity induces cell cycle re-entry, which is especially detrimental for survival of postmitotic neurons.

Regulation of neuronal cytoskeletal protein phosphorylation in neurodegenerative diseases.

Neuronal cytoskeletal proteins such as neurofilaments (NFs) and tau are aberrantly and hyperphosphorylated in neurodegeneration. Under normal physiological conditions, NFs are synthesized in the cell bodies and phosphorylated and transported in the axonal compartment. However, under neurodegenerative disorders such as Alzheimers disease (AD), spinal cord motor neuron inclusions of amyotrophic lateral sclerosis, Lewy bodies of Parkinsons disease, Picks disease, Charcot-Marie-Tooth disease, and diabetic neuropathy, NFs are aberrantly and hyperphosphorylated in cell bodies. The proline directed protein kinases, such as cyclin-dependent protein kinase 5, mitogen activated protein kinase, and glycogen synthase kinase 3β, and the non proline-directed kinases, such as casein kinase 1, are deregulated in AD. Moreover, the reversible phosphorylation by protein phosphatase, PP2A, which mainly carries out the dephosphorylation of tau and NFs, is down regulated in AD brain. The aberrant phosphorylation of cytoskeletal proteins such as tau and NFs results in the axonal transport defects in neurodegeneration. The peptidyl-prolyl isomerase Pin1 plays a regulatory role in the post-phosphorylation mechanism of neuronal cytoskeletal proteins in AD brain. Possible therapeutic interventions for neurodegenerative disorders are (1) inhibition of proline-directed kinases, (2) activation of protein phosphatases such as PP2A, and (3) modulation of peptidyl-prolyl isomerases such as Pin1. Here, I discuss the regulation of neuronal cytoskeletal proteins under physiology and pathology.

Phosphorylation of p27Kip1 at Thr187 by cyclin-dependent kinase 5 modulates neural stem cell differentiation.

Cdk5 plays a role in nervous system development; its role in the initial stages of neural differentiation is poorly understood. We isolated neural stem cells from E13 Cdk5 WT and KO mouse and observed them as they switched from proliferating stage to neural differentiation. We show that Cdk5 phosphorylation of p27kip1 at Thr187 is crucial to neural differentiation.

Regulation of Sox6 by Cyclin Dependent Kinase 5 in Brain

Cyclin dependent kinase 5 (Cdk5) is a proline-directed Ser/Thr kinase involved in various biological functions during normal brain development and neurodegeneration. In brain, Cdk5 activity is specific to post-mitotic neurons, due to neuronal specific expression of its activator p35. The biological functions of Cdk5 have been ascribed to its cytoplasmic substrates, however not much is known in nucleus. Here, we show that nuclear transcription factor Sox6 is a direct nuclear target of Cdk5. Sox6 is expressed in Tuj1 positive neurons, suggesting that Sox6 is expressed in differentiating neurons. The expression of Sox6 is high in mitotic nuclei during embryonic day 12 (E12) and gradually decreases during development into adult. On the other hand, Cdk5 expression gradually increases during its development. We show that Sox6 is expressed in mitotic nuclei in embryonic day 12 (E12) and in migrating neurons of E16. Sox6 is phosphorylated in vivo. Sox6 was detected by phospho-Ser/Thr and phospho-Ser/Thr-Pro and MPM-2 (Mitotic protein #2) antibodies in brain. Furthermore, calf intestinal alkaline phosphatase (CIAP) digestion resulted in faster migration of Sox6 band. The GST-Sox6 was phosphorylated by Cdk5/p35. The mass spectrometry analysis revealed that Sox6 is phosphorylated at T119PER motif. We show that Sox6 steady state levels are regulated by Cdk5. Cdk5 knockout mice die in utero and Sox6 protein expression is remarkably high in Cdk5−/− brain, however, there is no change in mRNA expression, suggesting a post-translational regulation of Sox6 by Cdk5. Transfection of primary cortical neurons with WT Cdk5 reduced Sox6 levels, while dominant negative (DN) Cdk5 and p35 increased Sox6 levels. Thus, our results indicate that Cdk5 regulates Sox6 steady state protein level that has an important role in brain development and function.

Topographic Regulation of Neuronal Intermediate Filament Proteins by Phosphorylation: In Health and Disease

Neurofilaments (NFs) belong to type 1 V family of intermediate filaments and are the most abundant proteins of the nervous system. Mammalian NF triplet proteins constitute three subunits, low molecular weight (NF-L), medium molecular weight (NF-M), and high molecular weight (NF-H) subunit proteins. The NF-M/H carboxy terminal tail domain has multiple KSP repeats (40≥100) depending upon species, the phosphorylation of which regulates axonal caliber and axonal transport. The NF tail domain contains phosphorylation sites for proline directed kinases such as mitogen activated protein kinases (MAPKs), cyclin dependent protein kinase 5 (Cdk5), c-Jun amino terminal kinase (JNKs), and glycogen synthase kinase-3 (GSK-3). The signaling cascades involved in NF phosphorylation like MAPKs and Cdk5 can also be affected by myelin associated glycoprotein (MAG). MAG activates the Erk1/2 and Cdk5 activities due to the glial/axon interaction and increases the proline directed Ser/Thr phosphorylation of C-terminal domain of NF-M/H. The NF phosphorylation is topographically regulated. In normal neurons, NFs are phosphorylated only in the axonal compartment. However, in degenerative neurons such as Alzheimer’s disease (AD), spinal cord motor neuron inclusions of amyotrophic lateral sclerosis (ALS), Lewy bodies of Parkinson’s disease (PD), and Pick’s disease neurofilament proteins are aberrantly phosphorylated in the cell bodies. Aberrant phosphorylation of NFs in neurodegeneration is either due to the deregulation of proline directed kinases such as MAPKs and Cdk5 or the downregulation of protein phosphatases such as protein phosphatase 2A (PP2A), calcineurin (PP2B), or both. Recently, studies from our laboratory have shown that peptidyl prolyl isomerase 1 (Pin1) stabilizes the NF phosphorylation in normal and stressed neurons. Pin1 selectively binds to the phosphorylated Ser/Thr-Pro residues and converts the cis isomers to the more stable trans isomers. The multiple SP repeats of NF-M/H are stabilized by Pin1 in a phosphorylation specific manner. Pin1 modulates the excitotoxic and oxidative stress induced perikaryal phosphorylation of NF-M/H. Here, we have discussed the factors regulating the topographic phosphorylation of NFs in health and disease.

Deregulation of Cytoskeletal Protein Phosphorylation and Neurodegeneration

Phosphorylation of cytoskeletal proteins is tightly regulated by the activities of multiple protein kinases and phosphatases. Three kinases, Cyclin-dependent kinase 5 (Cdk5), Glycogen synthase kinase 3β (GSK3β) and MAPKs have been implicated in their direct involvement in neuronal cytoskeletal protein phosphorylation. Cdk5 and GSK3β have been identified as prime candidates for pathogenesis. Cdk5 is a proline-directed serine/threonine protein kinase that requires an interaction with its activators, p35 or p39, to be catalytically active. While Cdk5 expression is ubiquitous, p35 and p39 are abundantly expressed in postmitotic neurons which, therefore, exhibit enhanced levels of Cdk5 activity. A pleiotropic kinase, Cdk5 has a multifunctional role in the mammalian central nervous system. Cdk5 was originally identified as a major Tau kinase. It associates with early stages of neurofibrillary tangles (NFTs). NFTs are composed mainly of hyperphosphorylated Tau aggregates, the pathological hallmarks of neurodegenerative tauopathies and Alzheimer’s disease (AD). Cdk5, by phosphorylating neuronal cytoskeletal proteins, such as Tau and neurofilaments (NFs), plays a critical role in neurodegeneration. In this review, we focus on the specific roles of Cdk5 phosphorylation of the neuronal cytoskeletal proteins (NFs and Tau) that contribute to neurodegeneration.