Proline-directed Kinases Research Articles

Abstract IA28: Immunological insights from tracking dynamic cellular behavior in lymph nodes and tissues

Abstract A unique feature of the vertebrate immune system is its dependency on the migratory capacity of individual immune cells for proper development, differentiation and functional execution in vivo. High-resolution dynamic live-animal imaging has allowed scientists to decipher novel immunological insights, which is aided by increasing imaging sophistication, availability of in vivo experimental models, and high throughput multi-parameter data collection and analyses. Intravital imaging, combined with mathematical modeling and other immunological tools, has revealed an asymmetric role for MHC interactions in modulating CD4+ vs. CD8+ T lymphocyte surveillance strategies for cognate antigens in lymph nodes (LNs) [1]. Furthermore, naïve CD8+ T cells are preferentially attracted in a CCR5-dependent manner to LN niche sites where cognate antigen recognition occurs between DCs and CD4+ T cells via CCL3 / CCL4 gradient [2]. Additional data suggest that naïve CD8+ T cells regulate transient expression of preformed inflammatory chemokine receptors via integrin engagement early upon entry into inflamed LNs, suggesting a context-specific orchestration of immune responses in LNs. Once activated in the LNs, T cells travel to various anatomic compartments for delivery of their acquired effector function. Recent imaging experiments revealed that extravascular, CNS-resident CX3CR1+ APCs dynamically extend their cellular processes across intact vessel wall into vascular lumen. The frequency of projections increased in experimental autoimmune encephalomyelitis (EAE), whereas the number of projections remained stable compared to baseline days after other tissue injury such as CNS tumor infiltration and aseptic spinal cord trauma [3]. This unique behavior by CNS parenchymal CX3CR1+ cells may represent their unexplored functional role in antigen sampling and immune cell recruitment across the BBB. Lastly, we reported a novel role for cyclin-dependent kinase 5 (Cdk5), a proline-directed serine-threonine kinase, in T cell activation and persistence [4]. Cdk5 enzymatic activity has been found to be aberrantly up-regulated in a wide spectrum of tumors. Disruption of Cdk5 activity sensitizes tumor cells to elimination in a T cell-dependent manner, a process that is associated with an attenuated response in PD-L1 up-regulation upon interferon-γ exposure. Our additional emerging data further support a synergistic role of anti-Cdk5 agents in modulating tumor sensitivity to immunotherapeutic approaches. In conclusion, the tissue microenvironment plays a critical role in regulating host immune cell tumor crosstalk, and recent new advances in imaging modalities and in vivo experimental methodologies provided new insights that hold promises to propel the field of cancer immunotherapy forward.

Proline Conformation in a Functional Tau Fragment

The conformational state of distinct prolines can determine the folding of a protein but equally other biological processes when coupled to a conformation-sensitive secondary reaction. For the neuronal tau protein, the importance of proline conformation is underscored by its interaction with different prolyl cis/trans isomerases. The proline conformation would gain even further importance after phosphorylation of the preceding residue by various proline-directed kinases. A number of molecular diseases including Alzheimer's disease and traumatic brain injury were thereby recently qualified as “cistauosis”, as they would imply a cis conformation for the pThr231-Pro232 prolyl bond. We here investigate by NMR spectroscopy the conformation of all prolines in a functional Tau fragment, Tau[208-324]. Although we can detect and identify some minor conformers in the cis form, we show that all prolines are for over 90% in the trans conformation. Phosphorylation by CDK2/CycA3, which notably leads to complete modification of the Thr231 residue, does not change this conclusion. Our data hence disagree with the notion that specific prolyl bonds in tau would adopt preferentially the cis conformation.

Analysis of the Inhibitory Elements in the p5 Peptide Fragment of the CDK5 Activator, p35, CDKR1 Protein.

Besides the hallmark pathology of amyloid plaques and neurofibrillary tangles, it is well documented that cyclin-dependent kinase 5 (CDK5), a critical neuronal protein kinase in nervous system development, function, and survival, when deregulated and hyperactivated induces Alzheimer’s disease (AD) and amyotrophic lateral sclerosis and Parkinson’s disease-like phenotypes in mice. In a recent study, we demonstrated that p5, a small, truncated fragment of 24 amino acid residues derived from the CDK5 activator protein 35 (NCK5A, p35), selectively inhibited deregulated CDK5 hyperactivity and ameliorated AD phenotypes in model mice. In this study, we identified the most inhibitory elements in the p5 peptide fragment. Each amino acid residue in p5 was systematically replaced with its homologous residues that may still be able to functionally substitute. The effects of these p5 peptide analogs were studied on the phosphotransferase activities of CDK5/p35, CDK5/p25, ERK1, and GSK3β. The mimetic p5 peptide (A/V substitution at the C-terminus of the peptide) in the sequence, KNAFYERALSIINLMTSKMVQINV (p5-MT) was the most effective inhibitor of CDK5 kinase activity of 79 tested mimetic peptides including the original p5 peptide, KEAFWDRCLSVINLMSSKMLQINA (p5-WT). Replacement of the residues in C-terminus end of the peptide affected CDK5 phosphotransferase activity most significantly. These peptides were strong inhibitors of CDK5, but not the related proline-directed kinases, ERK1 and GSK3β.

Multi-site phosphorylation regulates NeuroD4 activity during primary neurogenesis: a conserved mechanism amongst proneural proteins.

BackgroundBasic Helix Loop Helix (bHLH) proneural transcription factors are master regulators of neurogenesis that act at multiple stages in this process. We have previously demonstrated that multi-site phosphorylation of two members of the proneural protein family, Ngn2 and Ascl1, limits their ability to drive neuronal differentiation when cyclin-dependent kinase levels are high, as would be found in rapidly cycling cells. Here we investigate potential phospho-regulation of proneural protein NeuroD4 (also known as Xath3), the Xenopus homologue of Math3/NeuroM, that functions downstream of Ngn2 in the neurogenic cascade.ResultsUsing the developing Xenopus embryo system, we show that NeuroD4 is expressed and phosphorylated during primary neurogenesis, and this phosphorylation limits its ability to drive neuronal differentiation. Phosphorylation of up to six serine/threonine-proline sites contributes additively to regulation of NeuroD4 proneural activity without altering neuronal subtype specification, and number rather than location of available phospho-sites is the key for limiting NeuroD4 activity. Mechanistically, a phospho-mutant NeuroD4 displays increased protein stability and enhanced chromatin binding relative to wild-type NeuroD4, resulting in transcriptional up-regulation of a range of target genes that further promote neuronal differentiation.ConclusionsMulti-site phosphorylation on serine/threonine-proline pairs is a widely conserved mechanism of limiting proneural protein activity, where it is the number of phosphorylated sites, rather than their location that determines protein activity. Hence, multi-site phosphorylation is very well suited to allow co-ordination of proneural protein activity with the cellular proline-directed kinase environment.Electronic supplementary materialThe online version of this article (doi:10.1186/s13064-015-0044-8) contains supplementary material, which is available to authorized users.

Phosphorylation of ORF1p is required for L1 retrotransposition

Although members of the L1 (LINE-1) clade of non-LTR retrotransposons can be deleterious, the L1 clade has remained active in most mammals for ∼100 million years and generated almost 40% of the human genome. The details of L1-host interaction are largely unknown, however. Here we report that L1 activity requires phosphorylation of the protein encoded by the L1 ORF1 (ORF1p). Critical phospho-acceptor residues (two serines and two threonines) reside in four conserved proline-directed protein kinase (PDPK) target sites. The PDPK family includes mitogen-activated protein kinases and cyclin-dependent kinases. Mutation of any PDPK phospho-acceptor inhibits L1 retrotransposition. The phosphomimetic aspartic acid can restore activity at the two serine sites, but not at either threonine site, where it is strongly inhibitory. ORF1p also contains conserved PDPK docking sites, which promote specific interaction of PDPKs with their targets. As expected, mutations in these sites also inhibit L1 activity. PDPK mutations in ORF1p that inactivate L1 have no significant effect on the ability of ORF1p to anneal RNA in vitro, an important biochemical property of the protein. We show that phosphorylated PDPK sites in ORF1p are required for an interaction with the peptidyl prolyl isomerase 1 (Pin1), a critical component of PDPK-mediated regulation. Pin1 acts via isomerization of proline side chains at phosphorylated PDPK motifs, thereby affecting substrate conformation and activity. Our demonstration that L1 activity is dependent on and integrated with cellular phosphorylation regulatory cascades significantly increases our understanding of interactions between L1 and its host.

Identifying novel targets of oncogenic EGF receptor signaling in lung cancer through global phosphoproteomics.

Mutations in the epidermal growth factor receptor (EGFR) kinase domain occur in 10-30% of lung adenocarcinoma and are associated with tyrosine kinase inhibitor (TKI) sensitivity. We sought to identify the immediate direct and indirect phosphorylation targets of mutant EGFRs in lung adenocarcinoma. We undertook SILAC strategy, phosphopeptide enrichment, and quantitative MS to identify dynamic changes of phosphorylation downstream of mutant EGFRs in lung adenocarcinoma cells harboring EGFR(L858R) and EGFR(L858R/T790M) , the TKI-sensitive, and TKI-resistant mutations, respectively. Top canonical pathways that were inhibited upon erlotinib treatment in sensitive cells, but not in the resistant cells include EGFR, insulin receptor, hepatocyte growth factor, mitogen-activated protein kinase, mechanistic target of rapamycin, ribosomal protein S6 kinase beta 1, and Janus kinase/signal transducer and activator of transcription signaling. We identified phosphosites in proteins of the autophagy network, such as ULK1 (S623) that is constitutively phosphorylated in these lung adenocarcinoma cells; phosphorylation is inhibited upon erlotinib treatment in sensitive cells, but not in resistant cells. Finally, kinase-substrate prediction analysis from our data indicated that substrates of basophilic kinases from, AGC and Calcium and calmodulin-dependent kinase groups, as well as STE group kinases were significantly enriched and those of proline-directed kinases from, CMGC and Casein kinase groups were significantly depleted among substrates that exhibited increased phosphorylation upon EGF stimulation and reduced phosphorylation upon TKI inhibition. This is the first study to date to examine global phosphorylation changes upon erlotinib treatment of lung adenocarcinoma cells and results from this study provide new insights into signaling downstream of mutant EGFRs in lung adenocarcinoma. All MS data have been deposited in the ProteomeXchange with identifier PXD001101 (http://proteomecentral.proteomexchange.org/dataset/PXD001101).

Systematic investigation of hierarchical phosphorylation by protein kinase CK2.

Phosphorylation is a crucial regulatory mechanism governing cellular signal transduction pathways, and despite the large number of identified sites to date, most mechanistic studies remain focused on individual phosphorylation sites. This study is the first to systematically determine specific consensus sequences for hierarchical phosphorylation events. The results indicate that individual phosphorylation sites should not be studied in isolation, and that larger, multisite phosphorylation motifs may have profound impact on cellular signaling. This article is part of a Special Issue entitled: Protein dynamics in health and disease. Guest Editors: Pierre Thibault and Anne-Claude Gingras.

Abstract 1609: Proline-directed kinase signaling in human hepatocellular carcinomas developed on non-fibrotic liver

Abstract Ten to 40% hepatocellular carcinoma (HCC) arise on non-cirrhotic liver, including 5% on non-fibrotic liver (nfHCC). These tumors provide an interesting model for the analysis of the hepatocarcinogenesis pathways without the confounding factors associated with cirrhosis. Carcinogenesis has been often associated with the deregulation of phosphorylation-dependent signaling pathways. Quantitative phosphoproteomics recently allowed us to unravel the specific activation of proline-directed kinases (PDK) in nfHCC. Our aim was then to use medium throughput analytical tools (Alphascreen® and AMMP®) to evaluate the activation status of PDK dependent signaling pathways in a series of nfHCC samples. We have used a phosphoproteomics set comprising surgical samples from nfHCC (METAVIR F0 or F1) and samples of histologically normal livers. Proteins were extracted and analyzed for the activation status of select signaling pathways such as MAPK using both Alphascreen® and AMMP® technologies. Because MAPK are prototypic PDKs and are deregulated in many cancers, we analyzed their activation in a series of 25 nfHCC as compared to 6 non-tumor samples and found an overall increased activity in nfHCC samples. Moreover the phosphorylation status of Calnexin, a MAPK substrate found in the phosphoproteomics analysis was monitored and compared in nfHCC samples. In conclusion, we have identified phosphopeptide signatures in human samples of nfHCC that led to the identification of a deregulated PDK-dependent network in those tumors. The elucidation of the functional causes and consequences of such deregulation is ongoing. Citation Format: Saïd Taouji, Kristen Leong, Lee A. Beausang, Daniela Arma, Violaine Moreau, Charles Balabaud, Paulette Bioulac-Sage, Jessica Zucman-Rossi, Martin Latterich, Jean Rosenbaum, Eric Chevet. Proline-directed kinase signaling in human hepatocellular carcinomas developed on non-fibrotic liver. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1609. doi:10.1158/1538-7445.AM2014-1609

Abstract 1043: Distinctive roles of Smad3 C-tail and linker phosphorylation between cancer progression and suppression in TGF-beta1/Smad3 signal

Abstract Transforming growth factor B1 (TGF-B) is the prototype of a large family of pleiotropic polypeptide cytokines that regulate a multitude of physiological functions. The ability of TGF-B1/Smad3 signal to inhibit proliferation of epithelial, neuronal and hematopoietic cells from which most human neoplasms originate suggests a role of TGF-B in tumor suppression. Smad3, a key transcription factor in TGF-B signaling, functions as both a positive and negative regulator in carcinogenesis. TGF-B induces phosphorylation of Smad3 C-terminal SXS motif in its canonical pathway as well as the serine/threonine residues in the linker region. The linker region has been known to contain multiple phosphorylation sites for proline-directed kinases such as CDK, c-Jun, p38 MAPK, Erk and GSK-3B. Here we present that the blockade of Smad3 C-terminal phosphorylation by adenoviral dominant-negative mutant promotes tumorigenesis, but markedly inhibits metastasis to the lung as induced by tail-vein injection and orthotopic transplant of human and mouse breast cancer cell lines. Conversely, mutation of Smad3 linker phosphorylation sites greatly suppresses tumorigenesis, while it enhances metastatic dissemination to the lung. In vitro study revealed that the Smad3 linker region is constitutively phosphorylated in the cancer cells, and that the mutation of Smad3 linker phosphorylation sites enhances the TGF-B1-induced EMT, growth arrest and apoptosis, suggesting that the canonical TGF-B1 signal mediated through C-tail phosphorylation is modulated by negative inputs from the linker phosphorylation. Furthermore, the blockade of linker phosphorylation reduces the stem cell population following suppression of mammosphere formation and downregulation of embryonic transcription factors including Oct4, NANOG and Sox-2, and induces apoptosis by repressing an antiapoptotic protein Bcl-2, suggesting that the Smad3 linker phosphorylation retains function for the maintenance of cancer stemness and survival. These results indicate that oncogenic and cancer-suppressive TGF-B1/Smad3 signals are regulated by the site-specific phosphorylation of Smad3 protein, imparting a new aspect to anti-cancer therapies that target the TGF-B signal pathway. This work was supported by a National Research Foundation grant of Korea (2009-0081756) funded by the Korea government. Citation Format: Eunjin Bae, Akira Ooshima, Seong-Jin Kim. Distinctive roles of Smad3 C-tail and linker phosphorylation between cancer progression and suppression in TGF-beta1/Smad3 signal. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1043. doi:10.1158/1538-7445.AM2014-1043

Abstract 2964: The codon 47 Pro47Ser polymorphism influences p53 phosphorylation and apoptotic function

Abstract The p53 tumor suppressor protein is a central signaling protein in response to oncogenic, genotoxic and metabolic stress. Single nucleotide polymorphisms (SNPs) in p53 can have a marked impact on p53 tumor suppressor function, and have been associated with increased cancer risk. Amino acid 47 of p53 is a conserved residue, located within the second transactivation domain of this protein. In wild type p53, this residue is proline (P47, or WT). Importantly, this proline is critical for the ability of proline-directed kinases to phosphorylate p53 on the adjacent residue, serine 46. Phosphorylation of serine 46 of p53 greatly enhances the ability of p53 to transactivate certain target genes, and to induce apoptosis. In some Africans and African Americans, amino acid 47 of p53 encodes serine (S47) instead of proline. We have shown that the S47 polymorphism eliminates phosphorylation on serine 46, and reduces p53's ability to induce apoptosis by at least 3-fold. We have created mice that contain the humanized p53 gene (Hupki) for either the S47 or WT alleles of p53. In new data, we show that tissues from these mice have significantly decreased apoptosis after gamma radiation. We also show that apoptosis induced by cisplatin is markedly defective in S47 mice and cell lines. Further, MEFs from S47 mice have a 15-fold increase in IC50 for cisplatin, indicating that this polymorphism will impact the efficacy of cisplatin therapy in Africans and African Americans. Finally, we have identified two critical p53 target genes, GLS2 (metabolism) and NOXA (apoptosis), which show significantly impaired transactivation by S47. The long-term objective of this research is to further characterize the function of the S47 variant, and to identify the contribution of this variant to cancer risk and progression in Africans and African Americans. Citation Format: Matthew Jennis, Monica Hollstein, Maureen E. Murphy. The codon 47 Pro47Ser polymorphism influences p53 phosphorylation and apoptotic function. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2964. doi:10.1158/1538-7445.AM2014-2964

P.1.g.050 Receptor-mediated [35S]GTPgammaS binding in postmortem human brain membranes: optimisation of assay conditions for future studies

ERK1 and ERK2 are related protein-serine/threonine kinases that participate in the Ras-Raf-MEK-ERK signal transduction cascade. This cascade participates in the regulation of a large variety of processes including cell adhesion, cell cycle progression, cell migration, cell survival, differentiation, metabolism, proliferation, and transcription. MEK1/2 catalyze the phosphorylation of human ERK1/2 at Tyr204/187 and then Thr202/185. The phosphorylation of both tyrosine and threonine is required for enzyme activation. Whereas the Raf kinase and MEK families have narrow substrate specificity, ERK1/2 catalyze the phosphorylation of hundreds of cytoplasmic and nuclear substrates including regulatory molecules and transcription factors. ERK1/2 are proline-directed kinases that preferentially catalyze the phosphorylation of substrates containing a Pro-Xxx-Ser/Thr-Pro sequence. Besides this primary structure requirement, many ERK1/2 substrates possess a D-docking site, an F-docking site, or both. A variety of scaffold proteins including KSR1/2, IQGAP1, MP1, β-Arrestin1/2 participate in the regulation of the ERK1/2 MAP kinase cascade. The regulatory dephosphorylation of ERK1/2 is mediated by protein-tyrosine specific phosphatases, protein-serine/threonine phosphatases, and dual specificity phosphatases. The combination of kinases and phosphatases make the overall process reversible. The ERK1/2 catalyzed phosphorylation of nuclear transcription factors including those of Ets, Elk, and c-Fos represents an important function and requires the translocation of ERK1/2 into the nucleus by active and passive processes involving the nuclear pore. These transcription factors participate in the immediate early gene response. The activity of the Ras-Raf-MEK-ERK cascade is increased in about one-third of all human cancers, and inhibition of components of this cascade by targeted inhibitors represents an important anti-tumor strategy. Thus far, however, only inhibition of mutant B-Raf (Val600Glu) has been found to be therapeutically efficacious.

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 Alzheimer's disease (AD), spinal cord motor neuron inclusions of amyotrophic lateral sclerosis, Lewy bodies of Parkinson's disease, Pick's 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.

Physiological and pathological phosphorylation of tau by Cdk5

Hyperphosphorylation of microtubule-associated protein tau is one of the major pathological events in Alzheimer’s disease (AD) and other related neurodegenerative diseases, including frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17). Mutations in the tau gene MAPT are a cause of FTDP-17, and the mutated tau proteins are hyperphosphorylated in patient brains. Thus, it is important to determine the molecular mechanism of hyperphosphorylation of tau to understand the pathology of these diseases collectively called tauopathy. Tau is phosphorylated at many sites via several protein kinases, and a characteristic is phosphorylation at Ser/Thr residues in Ser/Thr-Pro sequences, which are targeted by proline-directed protein kinases such as ERK, GSK3β, and Cdk5. Among these kinases, Cdk5 is particularly interesting because it could be abnormally activated in AD. Cdk5 is a member of the cyclin-dependent kinases (Cdks), but in contrast to the major Cdks, which promote cell cycle progression in proliferating cells, Cdk5 is activated in post-mitotic neurons via the neuron-specific activator p35. Cdk5-p35 plays a critical role in brain development and physiological synaptic activity. In contrast, in disease brains, Cdk5 is thought to be hyperactivated by p25, which is the N-terminal truncated form of p35 and is generated by cleavage with calpain. Several reports have indicated that tau is hyperphosphorylated by Cdk5-p25. However, normal and abnormal phosphorylation of tau by Cdk5 is still not completely understood. In this article, we summarize the physiological and pathological phosphorylation of tau via Cdk5.

Post‐phosphorylation control of dopamine transporter by peptidyl prolyl cis‐trans isomerase PIN1 (803.6)

The rat dopamine transporter (DAT) is phosphorylated at the N‐terminus on T53, a site that is followed by proline, making it specific for proline‐directed kinases such as ERK. We found that amphetamine (AMPH) stimulates DAT T53 phosphorylation and mutating this site affects DA transport and AMPH‐stimulated efflux, suggesting a role for T53 phosphorylation in these processes. Phosphorylation of proline‐directed residues strongly impacts protein conformation by reducing the rate of cis‐trans isomerization of the phospho (p)‐S/T‐P peptide bond. This in turn slows the rate of p‐S/T‐P dephosphorylation by preventing actions of phosphatases. The peptidyl prolyl cis‐trans isomerase, PIN1, specifically isomerizes p‐S/T‐P motifs and catalyzes the return of the bond to the trans configuration allowing dephosphorylation to occur. To test for such activities on DAT we have used the PIN1‐specific inhibitor juglone. In both rat striatal synaptosomes and heterologous expression systems juglone treatment rapidly increased the p‐T53 level suggesting that PIN1 acts on DAT and regulates dephosphorylation of T53. In addition, juglone strongly stimulated [3H]DA efflux from rat striatal synaptosomes, supporting a mechanistic link between efflux and p‐T53. These results identify a novel mechanism for regulation of DA efflux and suggest PIN1 as an enzyme that functions in post‐phosphorylation control of DAT function.Grant Funding Source: Supported by NIDA R01 DA 13147

Cyclin-dependent kinase 5 phosphorylates and induces the degradation of ataxin-2

The expansion of a polyQ repeat within the ataxin-2 protein causes spinocerebellar ataxia type 2 (SCA2). However, neither the precise pathological mechanism nor the physiological functions of ataxin-2 are known. Ataxin-2 contains 47 (S/T)P sequences, which are targeted by proline-directed protein kinases such as the cyclin-dependent kinase 5 (Cdk5). We hypothesized that ataxin-2 is phosphorylated by Cdk5. In fact, phosphorylation of ataxin-2 by Cdk5–p25 was shown using two methods: in vitro32P labeling and electrophoretic mobility shift on Phos-tag SDS-PAGE. The fractionation of ataxin-2 into three portions, the N-terminal fragment (NF, amino acids 1–507), the middle fragment (MF, amino acids 508–905), and the C-terminal fragment (CF, amino acids 906–1313) showed that NF and MF were phosphorylated slightly and highly, respectively, by Cdk5–p25 when expressed in COS-7 cells. Cdk5-mediated phosphorylation induced the degradation of NF remarkably and MF moderately. Furthermore, toxic ataxin-2-41Q underwent proteasomal degradation after phosphorylation by Cdk5. These results suggest that Cdk5 controls the abundance of both normal and polyQ-expanded ataxin-2 protein in neurons, which implies that Cdk5 activity is a therapeutic approach for SCA2.

Divergent and convergent roles for kinases and phosphatases in neurofilament dynamics.

C-terminal neurofilament phosphorylation mediates cation-dependent self-association leading to neurofilament incorporation into the stationary axonal cytoskeleton. Multiple kinases phosphorylate the C-terminal domains of the heavy neurofilament subunit (NF-H), including cyclin-dependent protein kinase 5 (CDK5), mitogen-activated protein kinases (MAPKs), casein kinase 1 and 2 (CK1 and CK2) and glycogen synthase kinase 3β (GSK3β). The respective contributions of these kinases have been confounded because they phosphorylate multiple substrates in addition to neurofilaments and display extensive interaction. Herein, differentiated NB2a/d1 cells were transfected with constructs expressing GFP-tagged NF-H, isolated NF-H sidearms and NF-H lacking the distal-most 187 amino acids. Cultures were treated with roscovitine, PD98059, Li(+), D4476, tetrabromobenzotriazole and calyculin, which are active against CDK5, MKK1 (also known as MAP2K1), GSK3β, CK1, CK2 and protein phosphatase 1 (PP1), respectively. Sequential phosphorylation by CDK5 and GSK3β mediated the neurofilament-neurofilament associations. The MAPK pathway (i.e. MKK1 to ERK1/2) was found to downregulate GSK3β, and CK1 activated PP1, both of which promoted axonal transport and restricted neurofilament-neurofilament associations to axonal neurites. The MAPK pathway and CDK5, but not CK1 and GSK3β, inhibited neurofilament proteolysis. These findings indicate that phosphorylation of neurofilaments by the proline-directed MAPK pathway and CDK5 counterbalance the impact of phosphorylation of neurofilaments by the non-proline-directed CK1 and GSK3β.

Prolyl‐isomerase Pin1 controls normal and cancer stem cells of the breast

Mammary epithelial stem cells are fundamental to maintain tissue integrity. Cancer stem cells (CSCs) are implicated in both treatment resistance and disease relapse, and the molecular bases of their malignant properties are still poorly understood. Here we show that both normal stem cells and CSCs of the breast are controlled by the prolyl-isomerase Pin1. Mechanistically, following interaction with Pin1, Notch1 and Notch4, key regulators of cell fate, escape from proteasomal degradation by their major ubiquitin-ligase Fbxw7α. Functionally, we show that Fbxw7α acts as an essential negative regulator of breast CSCs' expansion by restraining Notch activity, but the establishment of a Notch/Pin1 active circuitry opposes this effect, thus promoting breast CSCs self-renewal, tumor growth and metastasis in vivo. In human breast cancers, despite Fbxw7α expression, high levels of Pin1 sustain Notch signaling, which correlates with poor prognosis. Suppression of Pin1 holds promise in reverting aggressive phenotypes, through CSC exhaustion as well as recovered drug sensitivity carrying relevant implications for therapy of breast cancers.

Cyclin-dependent Kinase 5 (Cdk5) Regulates the Function of CLOCK Protein by Direct Phosphorylation

Circadian rhythm is a biological rhythm governing physiology and behavior with a period of ∼24 h. At the molecular level, circadian output is controlled by a molecular clock composed of positive and negative feedback loops in transcriptional and post-translational processes. CLOCK is a transcription factor known as a central component of the molecular clock feedback loops generating circadian oscillation. Although CLOCK is known to undergo multiple post-translational modifications, the knowledge of their entities remains limited. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine-threonine kinase that is involved in various neuronal processes. Here, we report that Cdk5 is a novel regulator of CLOCK protein. Cdk5 phosphorylates CLOCK at the Thr-451 and Thr-461 residues in association with transcriptional activation of CLOCK. The Cdk5-dependent regulation of CLOCK function is mediated by alterations of its stability and subcellular distribution. These results suggest that Cdk5 is a novel regulatory component of the core molecular clock machinery.

Global analysis of the effects of the V2 receptor antagonist satavaptan on protein phosphorylation in collecting duct

Satavaptan (SR121463) is a vasopressin V2 receptor antagonist that has been shown to improve hyponatremia in patients with cirrhosis, congestive heart failure, and syndrome of inappropriate antidiuresis. While known to inhibit adenylyl cyclase-mediated accumulation of intracellular cyclic AMP and potentially recruit β-arrestin in kidney cell lines, very little is known regarding the signaling pathways that are affected by this drug. To this end, we carried out a global quantitative phosphoproteomic analysis of native rat inner medullary collecting duct cells pretreated with satavaptan or vehicle control followed by the V2 receptor agonist desmopressin (dDAVP) for 0.5, 2, 5, or 15 min. A total of 2,449 unique phosphopeptides from 1,160 proteins were identified. Phosphopeptides significantly changed by satavaptan included many of the same kinases [protein kinase A, phosphoinositide 3-kinase, mitogen-activated protein kinase kinase kinase 7 (TAK1), and calcium/calmodulin-dependent kinase kinase 2] and channels (aquaporin-2 and urea transporter UT-A1) regulated by vasopressin. Time course clustering and kinase motif analysis suggest that satavaptan blocks dDAVP-mediated activation of basophilic kinases, while also blocking dDAVP-mediated inhibition of proline-directed kinases. Satavaptan affects a variety of dDAVP-mediated processes including regulation of cell-cell junctions, actin cytoskeleton dynamics, and signaling through Rho GTPases. These results demonstrate that, overall, satavaptan acts as a selective V2 receptor antagonist and affects many of the same signaling pathways regulated by vasopressin. This study represents the first "systems-wide" analysis of a "vaptan"-class drug and provides a wealth of new data regarding the effects of satavaptan on vasopressin-mediated phosphorylation events.

Abstract A24: A possible role for GSK3β in epithelial ovarian cancer chemoresistance by epigenetic regulation

Abstract Ovarian cancers are highly heterogeneous and while chemotherapy is the preferred treatment many patients are intrinsically resistant or quickly develop resistance. Furthermore, all tumors that recur ultimately become resistant. Recent evidence suggests that epigenetic deregulation may be a key factor in the onset and maintenance of chemoresistance. We set out to identify epigenetically silenced genes that affect chemoresistance. The epigenomes of a total of 45 ovarian samples were analyzed to identify epigenetically altered genes that segregate with platinum response, and further filtered with expression data to identify genes that were suppressed. A tissue culture carboplatin resistance screen was utilized to functionally validate this set of candidate platinum resistance genes. Our screen correctly identified 19 genes that when suppressed altered the chemoresistance of the cells in culture. Of the genes identified in the screen we further characterized one gene, Glycogen Synthase Kinase 3 Beta (GSK3β), a proline-directed serine-threonine kinase, to determine if we could determine the mechanism by which it increased resistance. Preliminary results have indicated that the loss of GSK3b decreased the level of apoptosis in response to carboplatin. Furthermore, ovarian cell lines transcriptionally silenced for GSK3β are more invasive, have migratory ability, and display a transformed epithelial to mesenchymal (EMT) phenotype. Taken together, we provide the evidence of a role for GSK3β as an important epigenetic regulator of chemoresistance in ovarian cancer and hypothesize EMT as one of the underlying mechanisms. Citation Format: Noelle L. Cutter, Mena Yacoub, Robert D. Lucito, Nevenka Dimitrova, Elena Lum, Michael Vigliotti, Kazimierz Wrzeszczynski, Douglas A. Levine. A possible role for GSK3β in epithelial ovarian cancer chemoresistance by epigenetic regulation. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A24.