Phosphorylation of NuMA by Aurora‐A kinase in PC‐3 prostate cancer cells affects proliferation,survival, and interphase NuMA localization

Aurora‐A is a serine/threonine kinase that has oncogenic properties in vivo. The expression and kinase activity of Aurora‐A are up‐regulated in multiple malignancies. Aurora‐A is a key regulator of mitosis that localizes to the centrosome from the G2 phase through mitotic exit and regulates mitotic spindle formation as well as centrosome separation. Overexpression of Aurora‐A in multiple malignancies has been linked to higher tumor grade and poor prognosis through mechanisms that remain to be defined. Using an unbiased proteomics approach, we identified the protein nuclear mitotic apparatus (NuMA) as a robust substrate of Aurora‐A kinase. Using a small molecule Aurora‐A inhibitor in conjunction with a reverse in‐gel kinase assay (RIKA), we demonstrate that NuMA becomes hypo‐phosphorylated in vivo upon Aurora‐A inhibition. Using an alanine substitution strategy, we identified multiple Aurora‐A phospho‐acceptor sites in the C‐terminal tail of NuMA. Functional analyses demonstrate that mutation of three of these phospho‐acceptor sites significantly diminished cell proliferation. In addition, alanine mutation at these sites significantly increased the rate of apoptosis. Using confocal immunofluorescence microscopy, we show that the NuMA T1804A mutant mis‐localizes to the cytoplasm in interphase nuclei in a punctate pattern. The identification of Aurora‐A phosphorylation sites in NuMA that are important for cell cycle progression and apoptosis provides new insights into Aurora‐A function. J. Cell. Biochem. 114: 823–830, 2013. © 2012 Wiley Periodicals, Inc.     

γ‐Secretase Inhibition Induces Muscle Hypertrophy in a Notch‐Independent Mechanism

A wide variety of cellular processes and signaling events are regulated by the proteolytic enzyme γ‐secretase. Notch‐1 is one of the substrates of γ‐secretase and its role in the regulation of muscle differentiation has been well described. Importantly, besides Notch‐1, a number of proteins have been identified to undergo proteolysis by γ‐secretase. To date, the specific role of γ‐secretase during embryonic skeletal muscle differentiation has not been studied. Therefore, we address this question through the analysis of in vitro grown chick myogenic cells during the formation of multinucleated myotubes. The γ‐secretase inhibitor DAPT (N‐N[‐(3,5‐Difluorophenacetyl‐l ‐alanyl)]‐S‐328 phenylglycine‐t‐butyl‐ester) induces muscle hypertrophy. Knockdown of Notch‐1 using siRNA specific to chick shows no significant effect in myotube size, suggesting that γ‐secretase‐dependent effects on muscle hypertrophy in chick myogenic cells are Notch‐1‐independent. We also investigate the effects of γ‐secretase inhibition in the whole proteomic profile of chick myogenic cells. We identified 276 differentially expressed proteins from Label‐free proteomic approach. Data overview of interaction network obtained from STRING show that after γ‐secretase inhibition cells exhibited imbalance in protein metabolism, cytoskeleton/adhesion, and Sonic Hedgehog signaling. The collection of these results provides new insights into the role of γ‐secretase in skeletal muscle hypertrophy.     

Masking of Transmembrane‐Based Retention Signals Controls ER Export of γ‐Secretase

γ‐Secretase is critically involved in the Notch pathway and in Alzheimer's disease. The four subunits of γ‐secretase assemble in the endoplasmic reticulum (ER) and unassembled subunits are retained/retrieved to the ER by specific signals. We here describe a novel ER‐retention/retrieval signal in the transmembrane domain (TMD) 4 of presenilin 1, a subunit of γ‐secretase. TMD4 also is essential for complex formation, conferring a dual role for this domain. Likewise, TMD1 of Pen2 is bifunctional as well. It carries an ER‐retention/retrieval signal and is important for complex assembly by binding to TMD4. The two TMDs directly interact with each other and mask their respective ER‐retention/retrieval signals, allowing surface transport of reporter proteins. Our data suggest a model how assembly of Pen2 into the nascent γ‐secretase complex could mask TMD‐based ER‐retention/retrieval signals to allow plasma membrane transport of fully assembled γ‐secretase.     

“Activation of tissue tranglutsaminase by removal of carboxyl‐terminal peptides”

Tissue transglutaminase (TGC or TG2) functions as transglutaminase (cross‐linking), deamidase, kinase, and disulfide isomerase and its activities are implicated in the pathogenesis of several human diseases. Proteolytic activation of zymogens in the transglutaminase family is not unusual. Plasma transglutaminase (FXIIIa), epidermal transglutaminase (TG 3), transglutaminase‐5, and microbial transglutaminase (MTG) can be subjected to proteolysis from specific proteases to generate the active functional enzyme. In the present study, calcium or GTP was essential for activation of TGC cross‐linking activity by trypsin in membrane fractions from human RBC and was accompanied by the conversion of TGC (80 kDa) to a smaller TG form (55 kDa). While bacterially expressed TGC showed no activity, bacterial expression of C‐terminal domain deletion constructs with carboxy‐terminal ends ranging from lysine 464 (TG464) to glycine 480 (TG480) produced enzymes that were highly active in cross‐linking activity. The product of a construct with a coding region ended at proline 446 (TG446), which interrupted the calcium‐binding domain, exhibited weak cross‐linking activity. TG480 and TG512 were characterized by about 80% and 10%, respectively, of the cross‐linking activities of TG464. This may indicate that the longer the peptide after the calcium binding domain, the less the enzymatic activity expressed, possibly because the folding of such peptide which interfere with the calcium binding site or the catalytic site. Western analysis of MCF7 and T47D human breast cancer cells transfected with TGC showed TGC as a major protein and TG as a minor fragment. Incubation of lysate from transfected cells with serum resulted in the conversion of the TGC to TG, a condition that may be comparable to injury or wounds that lead to rapid enzymatic transamidation activation. J. Cell. Biochem. 112: 3469–3481, 2011. © 2011 Wiley Periodicals, Inc.     

Intracellular Itinerary of Internalised β‐Secretase,BACE1, and Its Potential Impact on β‐Amyloid Peptide Biogenesis

β‐Secretase (BACE1) cleavage of the amyloid precursor protein (APP) represents the initial step in the formation of the Alzheimer's disease associated amyloidogenic Aβ peptide. Substantive evidence indicates that APP processing by BACE1 is dependent on intracellular sorting of this enzyme. Nonetheless, knowledge of the intracellular trafficking pathway of internalised BACE1 remains in doubt. Here we show that cell surface BACE1 is rapidly internalised by the AP2/clathrin dependent pathway in transfected cells and traffics to early endosomes and Rab11‐positive, juxtanuclear recycling endosomes, with very little transported to the TGN as has been previously suggested. Moreover, BACE1 is predominantly localised to the early and recycling endosome compartments in different cell types, including neuronal cells. In contrast, the majority of internalised wild‐type APP traffics to late endosomes/lysosomes. To explore the relevance of the itinerary of BACE1 on APP processing, we generated a BACE1 chimera containing the cytoplasmic tail of TGN38 (BACE/TGN38), which cycles between the cell surface and TGN in an AP2‐dependent manner. Wild‐type BACE1 is less efficient in Aβ production than the BACE/TGN38 chimera, highlighting the relevance of the itinerary of BACE1 on APP processing. Overall the data suggests that internalised BACE1 and APP diverge at early endosomes and that Aβ biogenesis is regulated in part by the recycling itinerary of BACE1.     

Stabilization of the prostate‐specific tumor suppressor NKX3.1 by the oncogenic protein kinase Pim‐1 in prostate cancer cells

Loss of NKX3.1 is an early and consistent event in prostate cancer and is associated with increased proliferation of prostate epithelial cells and poor prognosis. NKX3.1 stability is regulated post‐translationally through phosphorylation at multiple sites by several protein kinases. Here, we report the paradoxical stabilization of the prostate‐specific tumor suppressor NKX3.1 by the oncogenic protein kinase Pim‐1 in prostate cancer cells. Pharmacologic Pim‐1 inhibition using the small molecule inhibitor CX‐6258 decreased steady state levels and half‐life of NKX3.1 protein but mRNA was not affected. This effect was reversed by inhibition of the 26S‐proteasome, demonstrating that Pim‐1 protects NKX3.1 from proteasome‐mediated degradation. Mass spectrometric analyses revealed Thr89, Ser185, Ser186, Ser195, and Ser196 as Pim‐1 phospho‐acceptor sites on NKX3.1. Through mutational analysis, we determined that NKX3.1 phosphorylation at Ser185, Ser186, and within the N‐terminal PEST domain is essential for Pim‐1‐mediated stabilization. Further, we also identified Lys182 as a critical residue for NKX3.1 stabilization by Pim‐1. Pim‐1‐mediated NKX3.1 stabilization may be important in maintaining normal cellular homeostasis in normal prostate epithelial cells, and may maintain basal NKX3.1 protein levels in prostate cancer cells. J. Cell. Biochem. 114: 1050–1057, 2013. © 2012 Wiley Periodicals, Inc.