Proline-mediated Proteasomal Degradation of the Prostate-specific Tumor Suppressor NKX3.1

Rao, Varsha; Guan, Bin; Mutton, Laura N.; Bieberich, Charles J.

doi:10.1074/jbc.m112.352823

Cited by 10 publications

(15 citation statements)

References 59 publications

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“…While NKX2-1 protein was clearly displayed in SU-DHL-5, NKX3-1 protein was not detectable in that cell line ( Fig. 1B,C ), consistent with post-transcriptional inhibition which was described recently [25]. For this reason we focused our work on regulation and function of homeobox gene NKX2-1, showing ectopic expression at the RNA and protein level in DLBCL cell line SU-DHL-5.…”

Section: Resultssupporting

confidence: 84%

Ectopic Expression of Homeobox Gene NKX2-1 in Diffuse Large B-Cell Lymphoma Is Mediated by Aberrant Chromatin Modifications

et al. 2013

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Homeobox genes encode transcription factors ubiquitously involved in basic developmental processes, deregulation of which promotes cell transformation in multiple cancers including hematopoietic malignancies. In particular, NKL-family homeobox genes TLX1, TLX3 and NKX2-5 are ectopically activated by chromosomal rearrangements in T-cell neoplasias. Here, using transcriptional microarray profiling and RQ-PCR we identified ectopic expression of NKL-family member NKX2-1, in a diffuse large B-cell lymphoma (DLBCL) cell line SU-DHL-5. Moreover, in silico analysis demonstrated NKX2-1 overexpression in 5% of examined DLBCL patient samples. NKX2-1 is physiologically expressed in lung and thyroid tissues where it regulates differentiation. Chromosomal and genomic analyses excluded rearrangements at the NKX2-1 locus in SU-DHL-5, implying alternative activation. Comparative expression profiling implicated several candidate genes in NKX2-1 regulation, variously encoding transcription factors, chromatin modifiers and signaling components. Accordingly, siRNA-mediated knockdown and overexpression studies confirmed involvement of transcription factor HEY1, histone methyltransferase MLL and ubiquitinated histone H2B in NKX2-1 deregulation. Chromosomal aberrations targeting MLL at 11q23 and the histone gene cluster HIST1 at 6p22 which we observed in SU-DHL-5 may, therefore, represent fundamental mutations mediating an aberrant chromatin structure at NKX2-1. Taken together, we identified ectopic expression of NKX2-1 in DLBCL cells, representing the central player in an oncogenic regulative network compromising B-cell differentiation. Thus, our data extend the paradigm of NKL homeobox gene deregulation in lymphoid malignancies.

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Section: Resultssupporting

confidence: 84%

Ectopic Expression of Homeobox Gene NKX2-1 in Diffuse Large B-Cell Lymphoma Is Mediated by Aberrant Chromatin Modifications

et al. 2013

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“…The relative amount of protein was evaluated by densitometry and normalized to a-tubulin control. Semi-logarithmic plots of log 10 -densitometric ratios versus time were plotted to determine protein half life as described previously (Rao et al, 2012). …”

Section: Methodsmentioning

confidence: 99%

Role of Methionine Adenosyltransferase α2 and β Phosphorylation and Stabilization in Human Hepatic Stellate Cell Trans‐Differentiation

Ramani

Donoyan

Tomasi

et al. 2015

Journal Cellular Physiology

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Myofibroblastic trans-differentiation of hepatic stellate cells (HSCs) is an essential event in the development of liver fibrogenesis. These changes involve modulation of key regulators of the genome and the proteome. Methionine adenosyltransferases (MAT) catalyze the biosynthesis of the methyl donor, S-adenosylmethionine (SAMe) from methionine. We have previously shown that two MAT genes, MAT2A and MAT2B (encoding MATα2 and MATβ proteins respectively), are required for HSC activation and loss of MAT2A transcriptional control favors its up-regulation during trans-differentiation. Hence MAT genes are intrinsically linked to the HSC machinery during activation. In the current study, we have identified for the first time, post-translational modifications in the MATα2 and MATβ proteins that stabilize them and favor human HSC trans-differentiation. Culture-activation of human HSCs induced the MATα2 and MATβ proteins. Using mass spectrometry, we identified phosphorylation sites in MATα2 and MATβ predicted to be phosphorylated by mitogen-activated protein kinase (MAPK) family members [ERK1/2, V-Raf Murine Sarcoma Viral Oncogene Homolog B1 (B-Raf), MEK]. Phosphorylation of both proteins was enhanced during HSC activation. Blocking MEK activation lowered the phosphorylation and stability of MAT proteins without influencing their mRNA levels. Silencing ERK1/2 or B-Raf lowered the phosphorylation and stability of MATβ but not MATα2. Reversal of the activated human HSC cell line, LX2 to quiescence lowered phosphorylation and destabilized MAT proteins. Mutagenesis of MATα2 and MATβ phospho-sites destabilized them and prevented HSC trans-differentiation. The data reveal that phosphorylation of MAT proteins during HSC activation stabilizes them thereby positively regulating trans-differentiation.

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“…NKX3.1 turnover is regulated by ubiquitination, but it is also proteolyzed by proteasomes independent of ubiquitination. This ubiquitin-independent degradation is mediated by a 21-amino acid sequence in its C-terminal region (35). The proline residue in the sequence is essential for its ubiquitin-independent degron activity.…”

Section: Discussionmentioning

confidence: 99%

“…Among them, we were interested in a ubiquitin-independent degron that is found in the C-terminal region of NKX3.1, a tumor repressor (42). The ubiquitin-independent degron sequence is composed of 21 amino acids with a PXL motif in the middle (35). p35 has a similar, but not identical, sequence in residues 240 -258 (Fig.…”

Section: Effect Of Cdk5mentioning

confidence: 99%

Two Degradation Pathways of the p35 Cdk5 (Cyclin-dependent Kinase) Activation Subunit, Dependent and Independent of Ubiquitination

Takasugi¹,

Minegishi²,

Asada³

et al. 2016

Journal of Biological Chemistry

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Cdk5 is a versatile protein kinase that is involved in various neuronal activities, such as the migration of newborn neurons, neurite outgrowth, synaptic regulation, and neurodegenerative diseases. Cdk5 requires the p35 regulatory subunit for activation. Because Cdk5 is more abundantly expressed in neurons compared with p35, the p35 protein levels determine the kinase activity of Cdk5. p35 is a protein with a short half-life that is degraded by proteasomes. Although ubiquitination of p35 has been previously reported, the degradation mechanism of p35 is not yet known. Here, we intended to identify the ubiquitination site(s) in p35. Because p35 is myristoylated at the N-terminal glycine, the possible ubiquitination sites are the lysine residues in p35. We mutated all 23 Lys residues to Arg (p35 23R), but p35 23R was still rapidly degraded by proteasomes at a rate similar to wild-type p35. The degradation of p35 23R in primary neurons and the Cdk5 activation ability of p35 23R suggested the occurrence of ubiquitin-independent degradation of p35 in physiological conditions. We found that p35 has the amino acid sequence similar to the ubiquitin-independent degron in the NKX3.1 homeodomain transcription factor. An Ala mutation at Pro-247 in the degron-like sequence made p35 stable. These results suggest that p35 can be degraded by two degradation pathways: ubiquitin-dependent and ubiquitin-independent. The rapid degradation of p35 by two different methods would be a mechanism to suppress the production of p25, which overactivates Cdk5 to induce neuronal cell death. Cyclin-dependent kinases (Cdks)2 are a family of Ser/Thr kinases that are activated by binding a regulatory subunit called cyclin. Most members of Cdks are expressed in proliferating cells to promote cell cycle progression (1). In contrast, Cdk5 is activated by p35 or p39 non-cyclin proteins, which are mainly expressed in post-mitotic neurons (2). Cdk5 is a versatile kinase that is involved in many neuronal activities, including neuronal cell layer formation, synaptic transmission, membrane trafficking, and neuron cell death (3). p35 and p39 appear to share common and/or distinct functions for Cdk5, with p35 being the predominant activator. This is shown by the phenotypes of knock-out (KO) mice; p35 KO mice display abnormal neural layers in the cerebral cortex (4), and p39 KO mice do not show apparent abnormalities, whereas p35 and p39 double KO mice are perinatal lethal with abnormal neural layers, as are the Cdk5 KO mice (5-7). To understand the precise function of Cdk5-p35 in various neuronal activities, it is important to reveal the regulation mechanism of Cdk5 activity.As well as being cell cycle Cdks, Cdk5 is a stable protein and is expressed more abundantly than p35 in neurons (8, 9). Therefore, Cdk5 activity is determined primarily by the available amount of activator protein p35, and the protein amounts of p35 are regulated by the balance between synthesis and degradation (2). Although the synthesis of p35 is stimulated by NGF or BDNF (10, 11), the...

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Proline-mediated Proteasomal Degradation of the Prostate-specific Tumor Suppressor NKX3.1

Cited by 10 publications

References 59 publications

Ectopic Expression of Homeobox Gene NKX2-1 in Diffuse Large B-Cell Lymphoma Is Mediated by Aberrant Chromatin Modifications

Ectopic Expression of Homeobox Gene NKX2-1 in Diffuse Large B-Cell Lymphoma Is Mediated by Aberrant Chromatin Modifications

Role of Methionine Adenosyltransferase α2 and β Phosphorylation and Stabilization in Human Hepatic Stellate Cell Trans‐Differentiation

Two Degradation Pathways of the p35 Cdk5 (Cyclin-dependent Kinase) Activation Subunit, Dependent and Independent of Ubiquitination

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