Retrospective assessment of cyclin-dependent kinase 5 mRNA and protein expression and its association with patient survival in breast cancer

Cyclin-dependent kinase 5 (Cdk5) is an atypical member of the cyclin-dependent kinase family and functions as a serine/threonine kinase that can be activated by non-cyclin binding activators p35 or p39. Cdk5 expression and activity has been linked with the development and progression of cancer; however, its expression in breast cancer has not been fully described. Protein expression of Cdk5 was determined in a large cohort of early-stage invasive breast cancer tumours (n = 1110) with long-term follow-up data using immunohistochemistry. Expression of CDK5 mRNA was assessed in the METABRIC cohort (n = 1980). Low nuclear and cytoplasmic expression of Cdk5 expression was significantly associated with shorter breast cancer-specific survival (P = .004 and P = .001, respectively). Importantly, low nuclear and cytoplasmic expression of Cdk5 remained associated with survival in multivariate analysis, including potentially confounding factors (hazard ratio (HR) = 0.612, 95% confidence interval (CI) = 0.418-0.896, P = .011 and HR = 0.507, 95% CI = 0.318-0.809, P = .004, respectively). In addition, low nuclear and cytoplasmic expression of Cdk5 was significantly associated with clinicopathological criteria associated with adverse patient prognosis. Low CDK5 mRNA expression was associated with shorter patient survival (P = .005) in the METABRIC cohort; no associations between copy gain or loss and survival were observed. These data suggest that low Cdk5 expression is associated with poor clinical outcome of breast cancer patients and may be of clinical relevance.     

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.     

Modulation of TRPA1 channel activity by Cdk5 in sensory neurons

Transient receptor potential cation channel, subfamily A, member 1 (TRPA1), is activated by a broad range of noxious stimuli. Cdk5, a member of the Cdk family, has recently been identified as a modulator of pain signaling pathways. In the current study, we investigated the extent to which Cdk5 modulates TRPA1 activity. Cdk5 inhibition was found to attenuate TRPA1 response to agonist in mouse DRG sensory neurons. Additionally, the presence of active Cdk5 was associated with increased TRPA1 phosphorylation in transfected HEK293 cells that was roscovitine-sensitive and absent in the mouse mutant S449A full-length channel. Immunopurified Cdk5 was observed to phosphorylate human TRPA1 peptide substrate at S448A in vitro. Our results point to a role for Cdk5 in modulating TRPA1 activity.     

Increased angiogenesis in Cdk4R24C/R24C:Apc+/Min intestinal tumors

Cyclin dependent kinase 4 (Cdk4) is a cell cycle regulator involved in early G1 cell cycle progression and has been indirectly implicated in angiogenesis in the Min mouse system, a mouse that harbors a mutation in the Apc gene. Apc^+/Min mice when crossed with Ink4a/arf-/- mice, exhibited increased angiogenesis of colorectal tumors suggesting that dysregulation of Cdk4 (due to loss of Ink4a-mediated suppression) may contribute to enhanced angiogenesis. To demonstrate a direct role for Cdk4 in angiogenesis, we crossed mice that have an activated Cdk4, Cdk4^R24C/R24C mice, with Apc^+/Min mice and examined levels of angiogenesis in intestinal tumors formed. Our results show an increase in the percentage of highly vascularized tumors in Cdk4^R24C/R24C:Apc^Min/+ and Cdk4^+/R24C:Apc^Min/+ mice compared to Cdk4^+/+:Apc^Min/+ mice. In addition immunohistochemical analysis showed an increase in CD-31 staining localized to endothelial cells of Cdk4^R24C/R24C:Apc^Min/+ mouse tumors, supporting the hypothesis of increased vasculature in these tumors. Further analysis showed an increase in the expression of the E2F1 target proteins Vegf-b and Cyclin A in Cdk4^R24C/R24C:Apc^+/Min intestinal tumors. Together these data suggest that the dysregulated Cdk4 gene plays an important role in angiogenesis during intestinal tumor formation and may in part act via increasing E2F1 target proteins. This is the first report to show that Cdk4 has a direct role in angiogenesis in vivo and may be an important drug target to reduce or prevent angiogenesis during intestinal tumor formation.     

Cdk5 is required for the positioning and survival of GABAergic neurons in developing mouse striatum

Cyclin‐dependent kinase 5 (Cdk5) is a serine/threonine kinase, and its activity is dependent upon an association with a neuron‐specific activating subunit. It was previously reported that Cdk5^−/− mice exhibit perinatal lethality and defective neuronal positioning. In this study, they focused on the analysis of neuronal positioning of GABAergic neurons in the forebrain. Defective formation of the ventral striatum, nucleus accumbens, and olfactory tubercles was found in Cdk5^−/− embryos. To further study this abnormal development, we generated and analyzed Dlx5/6‐Cre p35 conditional KO (cKO); p39^−/− mice in which forebrain GABAergic neurons have lost their Cdk5 kinase activity. Defective formation of the nucleus accumbens and olfactory tubercles as well as neuronal loss in the striatum of Dlx5/6‐Cre p35cKO; p39^−/− mice was found. Elevated levels of phosphorylated JNK were observed in neonatal striatal samples from Dlx5/6‐Cre p35cKO; p39^−/− mice, suggestive of neuronal death. These results indicate that Cdk5 is required for the formation of the ventral striatum in a cell‐autonomous manner, and loss of the kinase activity of Cdk5 causes GABAergic neuronal death in the developing mouse forebrain. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 419–437, 2017     

Transient induction of Cdk9 in the early stage of differentiation is critical for myogenesis

Cdk9 is a serine-threonine protein kinase that has been recognized as a regulator of cardiac differentiation. Recently, we have reported that transient induction of Cdk9 using noncoding RNA targeting Cdk9 sequences results in efficient cardiac differentiation. Concerning Cdk9 regulatory roles, here, we proposed whether constant overexpression of Cdk9 might influence the differentiation of myoblast C2C12 cells into myotubes. We overexpressed Cdk9 in mouse myoblast C2C12 cells to investigate its regulatory roles on myogenic differentiation. Upon Cdk9 overexpression, the expression level of myogenic regulatory factors was determined. Moreover, the expression profile of three important myomiRs consist of miR 1, 133 and 206 was examined during the differentiation process. Although Cdk9 expression is necessary for inducing differentiation in the early stage of myogenesis, continuous Cdk9 expression inhibits differentiation by modulating myomiRs and myogenic gene expression. Our results indicate that the transient induction of Cdk9 in the early stage of differentiation is critical for myogenesis.     

Bioinformatic survey for new physiological substrates of Cyclin-dependent kinase 5

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase predominantly active in the nervous system where it regulates several processes such as neuronal migration, cytoskeletal dynamics, axonal guidance, and neurotransmission.We constructed a position specific scoring matrix (PSSM) based on a dataset of sites shown to be phosphorylated both in vivo and in vitro by Cdk5. This dataset was curated manually through an exhaustive search of published experimental data. We then used this PSSM to perform a search in the mouse proteome through Scansite, a web-based tool for matching sequence patterns in large databases. Considering a stringent cut-off score of 0.5, we identified 354 new putative sites present in 291 proteins. In order to assess the robustness of our results, ten random subsets (of 80 sites each) of the original dataset were used to construct new PSSMs, which were then used as input for a new Scansite search, leading to the recovery of 81% of the 354 sites by at least 5 PSSMs.In order to reduce the number of false positives in our sequence-based approach, we evaluated which of these predicted sites were phosphorylated in vivo as determined by multiple phosphoproteomics studies carried out through mass spectrometry and available in the PhosphoSitePlus database. This step resulted in a very promising list of 132 putative phosphorylation sites for Cdk5, of which, 51 are specifically phosphorylated in brain tissue, and some are involved in functions regulated by Cdk5 such as axonal growth, synaptic plasticity and neurotransmission. Other phosphorylation sites in our list suggest that Cdk5 might regulate processes through mechanisms not previously recognized such as the control of mRNA splicing.     

Nucleocytoplasmic transfer of cyclin dependent kinase 5 and its binding to puromycin-sensitive aminopeptidase in <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis>

The Dictyostelium discoideum homolog of mammalian cyclin dependent kinase 5 (Cdk5) has previously been shown to be required for optimal growth and differentiation in this model organism, however, the subcellular localization of the protein has not previously been studied. In this study, immunolocalizations and a GFP fusion construct localized Cdk5 predominantly to the nucleus of vegetative cells. Western blots showed that Cdk5 was present in both nuclear and non-nuclear fractions, suggesting a functional role in both cellular locales. During the early stages of mitosis, Cdk5 gradually moved from a punctate nucleoplasmic distribution to localize adjacent to the inner nuclear envelope. During anaphase and telophase, Cdk5 localized to the cytoplasm and was not detected in the nucleoplasm. Cdk5 returned to the nucleus during cytokinesis. Proteolytic activity has been shown to be a critical regulator of the cell cycle. Immunoprecipitations coupled with immunolocalizations identified puromycin-sensitive aminopeptidase A (PsaA) as a potential Cdk5 binding partner in Dictyostelium. Immunoprecipitations also identified two phosphotyrosine proteins (35 and 18 kDa) that may interact with Cdk5 in vivo. Together, this work provides new insight into the localization of Cdk5, its function during cell division, and its binding to a proteolytic enzyme in Dictyostelium.     

Transfection with the E1A and E1B-19kDa Oncogenes Does Not Prevent Rat Embryo Fibroblasts from Cell-Cycle Arrest after γ-Irradiation

Introduction of the E1A early region of the human adenovirus type 5 impairs the ability of mammalian cells to stop in the cell cycle at G1/S after damage. Two-parameter fluorescence cell sorting with iododeoxyuridine revealed the radiation-induced G1/S arrest in rat embryo fibroblasts transformed with the complementing E1A and E1B-19kDa oncogenes. This was due to selective inhibition of CyclE/Cdk2-associated kinase activity, while activities of type 2 kinase and of CyclA/Cdk2 complexes remained unchanged. The inhibitor of G1-phase cyclin kinases, p21/Waf1, was accumulated and interacted with target kinases both in normal and in transformed cells after irradiation. As shown by immunoprecipitation, p21/Waf1 formed complexes with the E1A oncoproducts in the transformants, which possibly accounted for its functional inactivation. Kinase modification in cyclin–kinase complexes was assumed to play a key role in regulation of cyclin-dependent kinases in the transformants with inactivated p21/Waf1.     

High glucose increases Cdk5 activity in podocytes via transforming growth factor-β1 signaling pathway

Podocytes are highly specialized and terminally differentiated glomerular cells that play a vital role in the development and progression of diabetic nephropathy (DN). Cyclin-dependent kinase 5 (Cdk5), who is an atypical but essential member of the Cdk family of proline-directed serine/threonine kinases, has been shown as a key regulator of podocyte differentiation, proliferation and morphology. Our previous studies demonstrated that the expression of Cdk5 was significantly increased in podocytes of diabetic rats, and was closely related with podocyte injury of DN. However, the mechanisms of how expression and activity of Cdk5 are regulated under the high glucose environment have not yet been fully elucidated. In this study, we showed that high glucose up-regulated the expression of Cdk5 and its co-activator p35 with a concomitant increase in Cdk5 kinase activity in conditionally immortalized mouse podocytes in vitro. When exposed to 30 mM glucose, transforming growth factor-β1 (TGF-β1) was activated. Most importantly, we found that SB431542, the Tgfbr1 inhibitor, significantly decreased the expression of Cdk5 and p35 and Cdk5 kinase activity in high glucose-treated podocytes. Moreover, high glucose increased the expression of early growth response-1 (Egr-1) via TGF-β1-ERK1/2 pathway in podocytes and inhibition of Egr-1 by siRNA decreased p35 expression and Cdk5 kinase activity. Furthermore, inhibition of Cdk5 kinase activity effectively alleviated podocyte apoptosis induced by high glucose or TGF-β1. Thus, the TGF-β1-ERK1/2-Egr-1 signaling pathway may regulate the p35 expression and Cdk5 kinase activity in high glucose-treated podocytes, which contributes to podocyte injury of DN.     

Phosphorylation of adult type Sept5 (CDCrel-1) by cyclin-dependent kinase 5 inhibits interaction with syntaxin-1

Increasing evidence implicates cyclin-dependent kinase 5 (Cdk5) in neuronal synaptic function. We searched for Cdk5 substrates in synaptosomal fractions prepared from mouse brains. Mass spectrometric analysis after two-dimensional SDS-PAGE identified several synaptic proteins phosphorylated by Cdk5-p35; one protein identified was Sept5 (CDCrel-1). Although septins were isolated originally as cell division-related proteins in yeast, Sept5 is expressed predominantly in neurons and is implicated in exocytosis. We confirmed that Sept5 is phosphorylated by Cdk5-p35 in vitro and identified Ser17 of adult type Sept5 (Sept5_v1) as a major phosphorylation site. We found that Ser17 of Sept5_v1 is phosphorylated in mouse brains. Coimmunoprecipitation from synaptosomal fractions and glutathione S-transferase-syntaxin-1A pulldown assays of Sept5_v1 expressed in COS-7 cells showed that phosphorylation of Sept5_v1 by Cdk5-p35 decreases the binding to syntaxin-1. These results indicate that the interaction of Sept5 with syntaxin-1 is regulated by the phosphorylation of Sept5_v1 at Ser17 by Cdk5-p35.     

Structure and chromosomal localization of the mouse oncomodulin gene

The rat gene encoding oncomodulin (OM), a small calcium-binding protein, is under the control of a solo LTR derived from an endogenous intracisternal A-particle. The latter sequence is the only OM promoter analyzed so far. In order to study cell-type-specific OM expression in a species lacking LTR sequences in the OM locus, we initially synthesized an OM cDNA from mouse placenta. By sequencing, we found a 137-bp-long 5 leader region that differed markedly from its rat counterpart but had high similarity to several mouse genomic sequences. Primers specific to this sequence in addition with primers specific for an exon 2/intron 2 sequence were used to screen a mouse ES cell line genomic P1 library. One positive clone contained the whole OM gene, including intron 1 of 25 kb and a 5 flanking region of 27 kb lacking an LTR. The region upstream of exon 1 contains no TATA or CCAAT boxes but has a homopurine/homopyrimidine stretch of 102 bp as well as a (CA)₂₂ repeat. The latter sequence is polymorphic and was therefore, used to map the OM gene to the distal end of the long arm of mouse Chromosome (Chr) 5 by interspecific backcross analysis. Additonally we localized the OM gene by in situ hybridization to the region G1-3 on Chr 5, confirming the genetic linkage results. Finally, the OM gene was found to be structurally conserved and to exist in a single copy in mammals.     

A dual-color FISH gene map of the proximal region of rat Chromosome 4 and comparative analysis in human and mouse

The development and refinement of the rat genome map is a prerequisite for a continued qualified and fruitful use of this model system for the study of complex traits. In two distinct rat cancer models, recurrent amplification affecting the proximal region of rat Chr 4 was detected. To further characterize this region, we turned to the evolutionarily conserved chromosome segments in human Chr 7 and mouse Chrs 5 and 6 to identify functional and positional candidate genes. By means of single- and dual-color FISH on metaphase, prometaphase, and interphase chromatin, 15 genes in rat Chr 4q11-q23 (Cdk5, Hgf, Dmtf1, Abcb1, Cyp51, Cdk6, Tac1, Asns, Cav1, Met, Wnt2, Cftr, Smoh, Braf, Arhgef5) were mapped and aligned. In the course of this work, six cancer-related rat genes were isolated de novo and partly sequenced. Ten loci were also mapped by FISH in the mouse. The map provides the framework for a more detailed genetic characterization of individual tumor amplicons, but may also be valuable for the analysis of this region in other rat models of human complex disease. In addition, our data facilitate the analysis of events in mammalian chromosomal evolution affecting the region. In a comparison with human sequence data, we found that there is considerable conservation in this region both in gene order and in distances between genes. There is a single evolutionary breakpoint between rat and mouse and two between rat and human. Since our analysis shows that the three breaks all occurred in different positions, they must be independent of one another. The data tend to support the notion that the genomic configuration in rat Chr 4 is ancestral compared with that in humans and mice. Received: 7 June 2001 / Accepted: 7 August 2001     

ERK MAP Kinase Activation in Spinal Cord Regulates Phosphorylation of Cdk5 at Serine 159 and Contributes to Peripheral Inflammation Induced Pain/Hypersensitivity

Cyclin-dependent kinase 5 is a proline-directed serine/threonine kinase and its activity participates in the regulation of nociceptive signaling. Like binding with the activators (P35 or P25), the phosphorylation of Cdk5 plays a critical role in Cdk5 activation. However, it is still unclear whether Cdk5 phosphorylation (p-Cdk5) contributes to pain hyperalgesia. The aim of our current study was to identify the roles of p-Cdk5 and its upstream regulator in response to peripheral inflammation. Complete Freund''s adjuvant (CFA) injection induced acute peripheral inflammation and heat hyperalgesia, which was accompanied by sustained increases in phospho-ERK1/2 (p-ERK1/2) and phospho-Cdk5^S159 (p-Cdk5^S159) in the spinal cord dorsal horn (SCDH). CFA-induced p-ERK primarily colocalized with p-Cdk5^S159 in superficial dorsal horn neurons. Levels in p-ERK and p-Cdk5 were also increased in the 2^nd phase of hyperalgesia induced by formalin injection, which can produce acute and tonic inflammatory pain. MAP kinase kinase inhibitor U0126 intrathecal delivery significantly suppressed the elevation of p-Cdk5^S159, Cdk5 activity and pain response behavior (Heat hyperalgesia, Spontaneous flinches) induced by CFA or formalin injection. Cdk5 inhibitor roscovitine intrathecal administration also suppressed CFA-induced heat hyperalgesia and Cdk5 phosphorylation, but did not attenuate ERK activation. All these findings suggested that p-Cdk5^S159 regulated by ERK pathway activity may be a critical mechanism involved in the activation of Cdk5 in nociceptive spinal neurons contributes to peripheral inflammatory pain hypersensitivity.     

Cdk5 suppression blocks SIRT1 degradation via the ubiquitin-proteasome pathway in Parkinson's disease models

The NAD⁺-dependent protein deacetylase sirtuin 1 (SIRT1), a member of the sirtuin family, may have a neuroprotective effect in multiple neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD) and Amyotrophic lateral sclerosis (ALS). Many studies have suggested that overexpression-induced or resveratrol-treated activation of SIRT1 could significantly ameliorate several neurodegenerative diseases in mouse models. However, the type of SIRT1, protein expression levels and underlying mechanisms remain unclear, especially in PD. In this study, the results demonstrated that SIRT1 knockout markedly worsened the movement function in MPTP-lesioned animal model of PD. SIRT1 expression was found to be markedly decreased not only in environmental factor PD models, neurotoxin MPP⁺-treated primary culture neurons and MPTP-induced mice but also in genetic factor PD models, overexpressed α-synuclein-A30PA53T SH-SY5Y stable cell line and hm²α-SYN-39 transgenic mouse strain. Importantly, the degradation of SIRT1 during MPP⁺ treatment was mediated by the ubiquitin-proteasome pathway. Furthermore, the results indicated that cyclin-dependent kinase 5 (Cdk5) was also involved in the decrease of SIRT1 expression, which could be efficiently blocked by the inhibition of Cdk5. In conclusion, our findings revealed that the Cdk5-dependent ubiquitin-proteasome pathway mediated degradation of SIRT1 plays a vital role in the progression of PD.     

Cdk2 knockout mice are viable

BACKGROUND: Cyclin-dependent kinases (Cdks) and their cyclin regulatory subunits control cell growth and division. Cdk2/cyclin E complexes are thought to be required because they phosphorylate the retinoblastoma protein and drive cells through the G1/S transition into the S phase of the cell cycle. In addition, Cdk2 associates with cyclin A, which itself is essential for cell proliferation during early embryonic development. RESULTS: In order to study the functions of Cdk2 in vivo, we generated Cdk2 knockout mice. Surprisingly, these mice are viable, and therefore Cdk2 is not an essential gene in the mouse. However, Cdk2 is required for germ cell development; both male and female Cdk2(-/-) mice are sterile. Immunoprecipitates of cyclin E1 complexes from Cdk2(-/-) spleen extracts displayed no activity toward histone H1. Cyclin A2 complexes were active in primary mouse embryonic fibroblasts (MEFs), embryo extracts and in spleen extracts from young animals. In contrast, there was little cyclin A2 kinase activity in immortalized MEFs and spleen extracts from adult animals. Cdk2(-/-) MEFs proliferate but enter delayed into S phase. Ectopic expression of Cdk2 in Cdk2(-/-) MEFs rescued the delayed entry into S phase. CONCLUSIONS: Although Cdk2 is not an essential gene in the mouse, it is required for germ cell development and meiosis. Loss of Cdk2 affects the timing of S phase, suggesting that Cdk2 is involved in regulating progression through the mitotic cell cycle.