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. 2015;14(8):1327-36.
doi: 10.4161/15384101.2014.987621.

Cdk5 controls IL-2 gene expression via repression of the mSin3a-HDAC complex

Eric Lam  1 , Tej K Pareek, John J Letterio
Affiliations

Cdk5 controls IL-2 gene expression via repression of the mSin3a-HDAC complex

Eric Lam et al. Cell Cycle. 2015.

Abstract

Cyclin-dependent kinase 5 (Cdk5) is a unique member of a family of serine/threonine cyclin-dependent protein kinases. We previously demonstrated disruption of Cdk5 gene expression in mice impairs T-cell function and ameliorates T-cell-mediated neuroinflammation. Here, we show Cdk5 modulates gene expression during T-cell activation by impairing the repression of gene transcription by histone deacetylase 1 (HDAC1) through specific phosphorylation of the mSin3a protein at serine residue 861. Disruption of Cdk5 activity in T-cells enhances HDAC activity and binding of the HDAC1/mSin3a complex to the IL-2 promoter, leading to suppression of IL-2 gene expression. These data point to essential roles for Cdk5 in regulating gene expression in T-cells and transcriptional regulation by the co-repressor mSin3a.

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Figures

Figure 1.
Figure 1.
Cdk5 is required for optimal IL-2 expression. (A) Naïve wild type (Cdk5+/+) T cells or Cdk5-deficient (Cdk5−/-) T cells were activated with plate bound anti-CD3 and anti-CD28 antibodies for 48hrs in the presence or absence of Roscovitine. Amounts of IL-2 present in T cell supernatants were detected by ELISA. (B) Semi-quantitative RT-PCR analysis was performed to measure IL-2 mRNA expression in T cells activated by CD3/CD28 antibodies for 12hrs in the presence or absence of Roscovitine, and similarly, (C) IL-2 mRNA expression was determined in either Cdk5+/+ or Cdk5−/-) T cells following anti-CD3/CD28 activation.by semi-quantitative RT-PCR. (D) Summary diagram of IL-2 production after T cell activation with or without the presence of Cdk5 protein or Cdk5 activity.
Figure 2.
Figure 2.
Cdk5 interacts with HDAC1 and alters HDAC activity in activated (T)cells. (A) Immunoprecipitates of HDAC1 protein were prepared from lysates of T cells both before and after CD3/CD28 stimulation for 48 hrs. Precipitates were subjected to Western blot for both Cdk5 and HDAC1 protein. (B) The expression of HDAC1 mRNA and protein was examined in both naïve and activated T cells isolated from both wild type (Cdk5+/+) T cells and Cdk5-deficient (Cdk5−/-) T cells both before and after anti-CD3/CD28 stimulation. (C) HDAC activity was measured in both quiescent and anti-CD3/CD28 stimulated Cdk5+/+ and Cdk5−/- T cells. (D) Model depicting how Cdk5/HDAC1 forms a protein complex and inhibits HDAC activity. Cdk5-deficiency does not alter HDAC1 expression but does relieve the suppression of HDAC activity.
Figure 3.
Figure 3.
Cdk5 does not phosphorylate HDAC1 but does phosphorylate the mSin3a protein at serine residue 861. (A) Phosphorylation of HDAC1 by the Cdk5 kinase was measured by in vitro kinase assays. Cdk5 protein was immunoprecipitated from murine T cells and combined with either endogenous HDAC1 (End) isolated from murine T cells or with recombinant HDAC1 (Rec). Immunoprecipitates of Cdk5 from Cdk5−/- T cells were used as a negative (Neg) control. Neurofilament H (NF-H) is a known Cdk5 substrate and was used as a positive (Pos) control. (B) Phosphorylation of mSin3a by Cdk5 was determined using the previously described kinase assay, in which immunoprecipitates of Cdk5 isolated from murine T cells were incubated with either the endogenously expressed mSin3a (End) protein or recombinant mSin3a (Rec). (C) Schematic of mSin3a protein showing the amino acid sequence in the wild-type and mutant form of the protein used in these studies. In the mutant mSin3a, serine residue 861 was mutated to an alanine residue. (D) Wild-type or mutant forms of mSin3a were transfected into HEK293T cells and examined for expression using Western blot analysis. (E) Phosphorylation of Wild-type and mutant mSin3a protein was measured using a Cdk5 in vitro kinase assay. Immunoprecipitates of Cdk5 isolated from murine T cells were combined with either endogenous (End), Wild-type (WT) or mutant (MUT) mSin3a protein. Positive (Pos) and negative (Neg) controls were the same as described above. (F) Schematic diagram depicting how Cdk5 phosphorylates the mSin3a protein specifically at the Serin861 residue.
Figure 4.
Figure 4.
Cdk5 phosphorlyation of mSin3a at Serine861 disrupts the expression of the mSin3a protein. (A) RNA and protein expression for mSin3a were examined with semi-quantitative RT-PCR and Western blot analysis in wild type (Cdk5+/+) T cells or Cdk5−/- T cells with or without anti-CD3/CD28 activation. (B) mSin3a protein expression in either Cdk5+/+ or Cdk5−/- T cells treated with or without the proteasome inhibitor MG132 for 8 hrs. (C) Protein expression for mSin3a were examined in anti-CD3/CD28 activated Jurkat cells transfected with either Wild-type (WT) or mutant (MUT) mSin3a plasmids. Cells were treated in the presence or absence of the proteasome inhibitor MG132. (D) Schematic diagram depicting phosphorylation of the Serine861 residue on mSin3a by Cdk5 and the resultant proteasomal degradation of mSin3a.
Figure 5.
Figure 5.
Phosphorylation of mSin3a by Cdk5 disrupts the formation of the HDAC1/mSin3a complex. (A) HDAC1 immunoprecipitates were isolated from nuclear lysates of primary wild type (Cdk5+/+) T cells or Cdk5−/- T cells either before or after stimulation with anti-CD3/CD28 antibodies. Immunoprecipitates were subsequently probed for mSin3a and HDAC1 expression by Western blot. (B) ChIP analysis was performed to assess the binding of HDAC1 to the IL-2 promoter in either Cdk5+/+ or Cdk5−/- T cells, either before or after activation with anti-CD3/CD28 stimulation. (C) Similarly, ChIP analyses were performed on Jurkat cells transfected with either Wild-Type (WT) or mutant (MUT) mSin3a plasmids to determine the binding of HDAC1 to the IL-2 promoter. (D) Diagram depicting the disruption of HDAC1 occupancy of the IL-2 promoter upon TCR stimulation, due to the presence of Cdk5/p35 activity, and the persistence of HDAC1 on the IL-2 promoter when the expression / activity of Cdk5 is disrupted.
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References

    1. Dhavan R, Tsai LH. A decade of CDK5. Nat Rev Mol Cell Biol 2001; 2:749-59; PMID:11584302; http://dx.doi.org/10.1038/35096019 - DOI - PubMed
    1. Arif A. Extraneuronal activities and regulatory mechanisms of the atypical cyclin-dependent kinase Cdk5. Biochem Pharmacol 2012; 84:985-93; PMID:22795893; http://dx.doi.org/10.1016/j.bcp.2012.06.027 - DOI - PubMed
    1. Jessberger S, Gage FH, Eisch AJ, Lagace DC. Making a neuron: Cdk5 in embryonic and adult neurogenesis. Trends Neurosci 2009; 32:575-82; PMID:19782409; http://dx.doi.org/10.1016/j.tins.2009.07.002 - DOI - PMC - PubMed
    1. Pareek TK, Lam E, Zheng X, Askew D, Kulkarni AB, Chance MR, Huang AY, Cooke KR, Letterio JJ. Cyclin-dependent kinase 5 activity is required for T cell activation and induction of experimental autoimmune encephalomyelitis. J Exp Med 2010; 207:2507-19; PMID:20937706; http://dx.doi.org/10.1084/jem.20100876 - DOI - PMC - PubMed
    1. Nohara A, Okada S, Ohshima K, Pessin JE, Mori M. Cyclin-dependent kinase-5 is a key molecule in tumor necrosis factor-alpha-induced insulin resistance. J Biol Chem 2011; 286:33457-65; PMID:21813649; http://dx.doi.org/10.1074/jbc.M111.231431 - DOI - PMC - PubMed

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