Anton Chestukhin

Processing, localization, and requirement of human separase for normal anaphase progression

In all eukaryotes, anaphase is triggered by the activation of a protease called separase. Once activated, separase cleaves a subunit of cohesin, a complex that links replicated chromatids before anaphase. Separase and cohesin are conserved from yeasts to humans. Although the machinery for dissolving sister cohesion is conserved, the regulation of this process appears to be more complex in higher eukaryotes than in yeast. Here we report the cloning of full-length human separase cDNA and the characterization of the encoded protein. Human separase was observed at the poles of the mitotic spindle until anaphase, at which time its association with the mitotic spindle was abruptly lost. The dynamic pattern of localization of human separase during cell cycle progression differs from that of fungal separases. Human separase also appears to undergo an autocatalytic processing on anaphase entry. The processed forms of human separase were isolated and the identity of the cleavage sites was determined by N-terminal sequencing and site-directed mutagenesis. The processed catalytic domain was found to be stably associated with the processed N-terminal fragment. Finally, by depletion of endogenous separase with antisense oligonucleotides, we report direct evidence that separase is required for high-fidelity chromosome separation in human cells.

Nucleocytoplasmic Shuttling of p130/RBL2: Novel Regulatory Mechanism

ABSTRACT The retinoblastoma-related pocket proteins pRb, p107, and p130 are implicated in the control of cell proliferation, differentiation, and transformation. The function of pocket proteins is in part mediated by their ability to inhibit specific E2F transcription factors. The transcriptional activity of E2Fs is controlled by alteration of their nucleocytoplasmic localization during the cell cycle. p130 was observed to shuttle between the nucleus and cytoplasm in a heterokaryon fusion assay, suggesting the presence of nuclear and cytoplasmic localization signals. Two independent nuclear localization signals (NLS) that could target reporter proteins to the nucleus in transient transfection and microinjection experiments were identified in the C terminus of p130. In addition to the C-terminal NLS, the intact pocket domain of p130 itself was sufficient for nuclear translocation. Moreover, an additional functional NLS was mapped within the unique Loop region of p130. An N-terminal domain that conferred cytoplasmic localization was identified. Removal of the entire N terminus did not affect the ability of p130 to interact with E2F and to induce growth arrest. A model suggesting that the activity of pRb family members can be regulated by intracellular trafficking of the proteins is proposed.

Glycogen Synthase Kinase 3 Phosphorylates RBL2/p130 during Quiescence

ABSTRACT Phosphorylation of the retinoblastoma-related or pocket proteins RB1/pRb, RBL1/p107, and RBL2/p130 regulates cell cycle progression and exit. While all pocket proteins are phosphorylated by cyclin-dependent kinases (CDKs) during the G1/S-phase transition, p130 is also specifically phosphorylated in G0-arrested cells. We have previously identified several phosphorylated residues that match the consensus site for glycogen synthase kinase 3 (GSK3) in the G0 form of p130. Using small-molecule inhibitors of GSK3, site-specific mutants of p130, and phospho-specific antibodies, we demonstrate here that GSK3 phosphorylates p130 during G0. Phosphorylation of p130 by GSK3 contributes to the stability of p130 but does not affect its ability to interact with E2F4 or cyclins. Regulation of p130 by GSK3 provides a novel link between growth factor signaling and regulation of the cell cycle progression and exit.

Unveiling the substrate specificity of meprin beta on the basis of the site in protein kinase A cleaved by the kinase splitting membranal proteinase.

The kinase splitting membranal proteinase (KSMP) is a metalloendopeptidase that inactivates the catalytic (C) subunit of protein kinase A (PKA) by clipping off its carboxyl terminal tail. Here we show that this cleavage occurs at Glu332-Glu333, within the cluster of acidic amino acids (Asp328-Glu334) of the kinase. The Km values of KSMP and of meprin β (which reproduces KSMP activity) for the C-subunit are below 1 μM. The Km for peptides containing a stretch of four Glu residues are in the micromolar range, illustrating the significant contribution of this cluster to the substrate recognition of meprin β. This conclusion is supported by a systematic study using a series of the C-subunit mutants with deletions and mutations in the cluster of acidics. Hydrophobic amino acids vicinal to the cleavage site increase the Kcat of the proteinase. These studies unveil a new specificity for meprin β, suggesting new substrates that are 1-2 orders of magnitude better in their Km and Kcat than those commonly used for meprin assay. A search for substrates having such a cluster of acidics and hydrophobics, which are accessible to meprin under physiological conditions, point at gastrin as a potential target. Indeed, meprin β is shown to cleave gastrin at its cluster of five glutamic acid residues and also at the M-D bond within its WMDF-NH2 sequence, which is indispensable for all the known biological activities of gastrins. The latter meprin cleavage will lead to the inactivation of gastrin and thus to the control of its activity.

Phosphorylation of the retinoblastoma-related protein p130 in growth-arrested cells

The retinoblastoma family of proteins including pRB, p107 and p130 undergoes cell cycle dependent phosphorylation during the mid-G1 to S phase transition. This phosphorylation is dependent upon the activity of cyclin D/cdk4. In contrast to pRB and p107, p130 is phosphorylated during G0 and the early G1 phase of the cell cycle. We observed that p130 is specifically phosphorylated on serine and threonine residues in T98G cells arrested in G0 by serum deprivation or density arrest. Identification of the phospho-serine and phospho-threonine residues revealed that most were clustered within a short co-linear region unique to p130, defined as the Loop. Deletion of the Loop region resulted in a change in the phosphorylation status of p130 under growth arrest conditions. Notably, deletion of the Loop did not affect the ability of p130 to bind to E2F-4 or SV40 Large T antigen, to induce growth arrest in Saos-2 cells, and to become hyperphosphorylated during the proliferative phase of the cell cycle. p130 undergoes specific G0 phosphorylation in a manner that distinguishes it from pRB and p107.

THE CLEAVAGE OF PROTEIN KINASE A BY THE KINASE-SPLITTING MEMBRANAL PROTEINASE IS REPRODUCED BY MEPRIN BETA

The Kinase-Splitting Membranal Proteinase (KSMP) is a metallo-endoproteinase that clips off the carboxyl terminus tail of the catalytic (C) subunit of protein kinase A to yield a truncated, catalytically inactive protein (C′). Here we report (a) a new procedure for the purification of KSMP, yielding a major protein band in SDS-polyacrylamide gel electrophoresis that correlates with the characteristic KSMP activity; (b) the sequence of tryptic peptides obtained from this band, suggesting an identity between this protein and meprin β; (c) the immuno-detection by specific anti-peptide antibodies of both the α and the β subunits of meprin in KSMP preparations; (d) the stable expression of meprin β in a mammalian cell line (293) to establish a clone that constitutively expresses the full-length precursor of meprin β; and (e) the optimalization of the proteolytic activation of this precursor to obtain an enzyme exhibiting the specific KSMP cleavage, suggesting that KSMP is either derived from, or identical with, the meprin β gene product. It is hoped that these results will shed light on the possible physiological role of KSMP and the way it may affect protein kinase A-mediated processes.

Localization of protein kinase A and vitronectin in resting platelets and their translocation onto fibrin fibers during clot formation

Physiological stimulation of platelets with thrombin brings about the release of protein kinase A (PKA) into the plasma. In human blood, this kinase singles out and phosphorylates vitronectin (Vn), a multifunctional regulatory protein, which was proposed to play an important role in the control of fibrinolysis. Here we present immuno-cytochemical evidence to show: (i) that intact platelets possess on their surface an ecto-PKA which can preferentially phosphorylate Vn; (ii) that in the resting platelet, both the catalytic and the regulatory subunits of PKA are present on the platelet surface, in the surface-connected canalicular system, and within the alpha-granules of the platelets; (iii) that the process initiated upon platelet activation, which leads to the formation of fibrin fibers and consequently forms the fibrin net, is accompanied by a translocation of PKA, of Vn, and of PAI-1 onto the fibrin fibers. We propose that the localization and the translocation of these proteins in the fibrin net, together with our finding that PKA phosphorylation of Vn reduces its grip of PAI-1, can unleash PAI-1 in its free form. The free PAI-1 can then assume its latent (non inhibitory) conformation, allow plasminogen activators to trigger the formation of active plasmin, and to initiate fibrinolysis.

The carboxyl-terminal tail of kinase splitting membranal proteinase/meprin beta is involved in its intracellular trafficking.

The kinase splitting membranal proteinase (KSMP), was recently shown to be identical with the β-subunit of meprin. Meprin is a metalloendoproteinase located in brush border membranes and composed of the two types of subunits, α and β. Despite their high sequence homology and similar domain organization, meprin subunits are differently processed during maturation; meprin α is retained in the endoplasmic reticulum (ER), and undergoes a proteolytic removal of the transmembrane and cytoplasmic domains, prior to its export from this organelle. In contrast, meprin β retains these domains even after reaching its final destination in the plasma membrane. Using truncated mutants of rat meprin β expressed in Cos-7 and human embryonic kidney (HEK) 293 cells, we show here that the cytoplasmic tail is indispensable for its exit from the ER. A meprin β mutant lacking the last 25 amino acids is shown to be transport-incompetent, although it does not contain any of the known ER retention signals. Systematic analysis of the rate of the ER to Golgi transport using a series of mutants with Ala or Pro substitutions in the tail, suggests that while no specific amino acid residue by itself is imperative for normal intracellular trafficking of meprin β, the insertion of a bend at a distinct position in the tail (specifically by a Y685P mutation) suffices to retain this protein in the ER. We propose that the very length of the cytoplasmic tail, as well as its secondary structure are essential for the ER to Golgi transport of meprin β, possibly by allowing an interaction with a cargo receptor.

Anti-head and anti-tail antibodies against distinct epitopes in the catalytic subunit of protein kinase A Use in the study of the kinase splitting membranal proteinase KSMP

Protein kinases share a considerable sequence homology in their catalytic core (residues 40–300 in PKA). Each core is flanked by “head” and “tail” segments at its amino‐ and carboxy‐termini, which are different in the various kinases. These end segments may play an important role in creating the preferential affinity of each kinase for its physiological substrates or regulatory ligands. Here we describe three anti‐peptide antibodies (αP‐1, αP‐2, and αP‐3) that specifically recognize the head and tail segments of the catalytic subunit (C) of PKA. (i) αP‐1 (against 6A‐K23) react with C when denatured but not when in its native structure; (ii) αP‐2 (against 319K‐I335), bind to the site in C cleaved by the kinase splitting membranal proteinase (KSMP) and inhibit this cleavage of C; (iii) αP‐3 (against 338S‐F350) react with C but not with the KSMP cleavage product C′, useful for detecting a KSMP‐like activity in different tissues and subcellular loci. The combined use of the antibodies described here provides a strict definition of C, and thus a high degree of fidelity in its biorecognition.

Western blot screening for monoclonal antibodies against human separase

Separase is a cysteine protease that participates in separation of sister chromatids during mitosis. Human separase is a 230-kDa enzyme that is inhibited by binding to its protein inhibitor securin, specific phosphorylation, and subcellular localization. To further characterize human separase, we raised monoclonal antibodies specific against a C-terminal fragment of the protein. A critical step in monoclonal antibody production procedure is the primary screening of hybridoma supernatants. Here we report primary screening protocol utilizing Western blot analysis. The described screening protocol is carried out using fusion of a human separase fragment with two different purification tags, maltose-binding protein (MBP) and glutathione S-transferase (GST). Immunization by MBP-fusion was followed by primary screening with both MBP- and GST-separase fusions combined in the same preparation separated in SDS-PAGE. This highly sensitive screening approach reduced the number of positive signals by eliminating antibodies specific for the purification tag used in the immunization procedure. The described separase-specific antibodies were suitable for detection of endogenous separase in crude extracts, immunoprecipitation, and immunofluorescent cell staining experiments. The presented procedure is fast, reproducible and could be adopted as a primary screening scheme for a variety of protein antigens.