VLA-5-mediated Adhesion to Fibronectin Accelerates Hemin-stimulated Erythroid Differentiation of K562 Cells through Induction of VLA-4 Expression

Fibronectin plays important roles in erythropoiesis through the fibronectin receptors VLA-4 and VLA-5. However, the substantial role of these fibronectin receptors and their functional assignment in erythroid differentiation are not yet fully understood. Here, we investigated the effects of cell adhesion to fibronectin on erythroid differentiation using K562 human erythroid progenitor cells. Erythroid differentiation could be induced in K562 cells in suspension by stimulating with hemin. This hemin-stimulated erythroid differentiation was highly accelerated when cells were induced to adhere to fibronectin by treatment with TNIIIA2, a peptide derived from tenascin-C, which has recently been found to induce β1-integrin activation. Another integrin activator, Mn²⁺, also accelerated hemin-stimulated erythroid differentiation. Adhesive interaction with fibronectin via VLA-4 as well as VLA-5 was responsible for acceleration of the hemin-stimulated erythroid differentiation in response to TNIIIA2, although K562 cells should have been lacking in VLA-4. Adhesion to fibronectin forced by TNIIIA2 causally induced VLA-4 expression in K562 cells, and this was blocked by the RGD peptide, an antagonist for VLA-5. The resulting adhesive interaction with fibronectin via VLA-4 strongly enhanced the hemin-stimulated activation of p38 mitogen-activated protein kinase, which was shown to serve as a signaling molecule crucial for erythroid differentiation. Suppression of VLA-4 expression by RNA interference abrogated acceleration of hemin-stimulated erythroid differentiation in response to TNIIIA2. Thus, VLA-4 and VLA-5 may contribute to erythropoiesis at different stages of erythroid differentiation.Hematopoietic stem and progenitor cells proliferate and differentiate in the bone marrow and fetal liver (1–6). Stromal cells of the bone marrow and fetal liver form a hematopoietic microenvironment called a “niche.” This microenvironment niche plays a crucial role in the regulation of the proliferation and differentiation of hematopoietic stem and progenitor cells. Besides humoral factors that include hematopoietic growth factors, adhesive interaction of hematopoietic stem and progenitor cells with stromal cells and/or the extracellular matrix (ECM)² in the hematopoietic microenvironment is indispensable for hematopoietic development (1–6). The ECM in the hematopoietic microenvironment is composed of various macromolecules, such as fibronectin (FN), collagens, laminins, and proteoglycans. Among them, FN is one of the most important parts of the microenvironment niche (7–11). Also, in erythropoiesis, the importance of the adhesion of erythroid progenitors to FN via the FN receptors VLA-4 and VLA-5 has been reported (11–16). However, the substantial role of these FN receptors and their functional assignment in erythroid differentiation are not yet fully understood.We previously found that FN, which provides scaffolding for the adhesion of various cell types, has an alternative functional site opposing cell adhesion (17). A 22-mer peptide derived from the 14th FN type III-like (FNIII) repeat of the FN molecule, termed FNIII14, strongly suppresses cell adhesion to FN by inhibiting the activation of β1-integrins including VLA-4 and VLA-5 (18, 19). Conversely, we have recently found that tenascin (TN)-C, which is an anti-adhesive ECM protein (20, 21), has a functional site for stimulating cell adhesion to FN (22). A 22-mer peptide derived from the FNIII repeat A2 in the TN-C molecule, termed TNIIIA2, can induce the conformational change necessary for functional activation of FN receptors through binding with syndecan-4 (22, 23). The active sites of FNIII14 and TNIIIA2 appear to be cryptic in the molecular structures of FN and TN-C but are exposed by conformational change through interaction with other ECM molecules or by processing with matrix metalloproteinase-2 (22, 24). Thus, these functional sites found in FN and TN-C molecules, which act in opposition to their parental ECM proteins, may act as a negative feedback loop for preventing excessive cellular responses to these ECM proteins in biological processes with ECM rearrangement. In any case, FNIII14 and TNIIIA2 enable us to control, either negatively or positively, the adhesion of various cell types to FN.Various hematopoietic progenitor cell lines have been used in in vitro studies of hematopoietic differentiation. However, most hematopoietic progenitor cell lines are nonadherent, because their cell surface β1-integrins, including FN receptors, have impaired ligand-binding activity (25, 26). Therefore, in order to investigate the role of cell adhesion to FN in hematopoietic differentiation, their FN receptors must be activated. Since TNIIIA2 can induce activation of FN receptors in various hematopoietic progenitor cell lines (22), this peptide factor may be useful for investigating the substantial role of cell adhesion to FN in hematopoietic differentiation. Here, we investigate the effects of cell adhesion to FN on erythroid differentiation using TNIIIA2 and Mn²⁺ as the integrin activator and the human erythroid progenitor cell line K562, which only expresses VLA-5, as the FN receptor (27). As a result, we show that hemin-stimulated erythroid differentiation of K562 cells is strongly enhanced when K562 cells are forced to adhere to FN. Sustained adhesion to FN via VLA-5, which is induced by TNIIIA2 or Mn²⁺, causes induction of VLA-4 expression. The resulting adhesive interaction with FN via newly expressed VLA-4 then generates a conspicuous increase in the hemin-stimulated phosphorylation/activation of p38 MAP kinase, which is shown to serve as a signaling molecule crucial for erythroid differentiation of K562 cells.     

Identification of Lysine Succinylation Substrates and the Succinylation Regulatory Enzyme CobB in Escherichia coli

Lysine succinylation is a newly identified protein post-translational modification pathway present in both prokaryotic and eukaryotic cells. However, succinylation substrates and regulatory enzyme(s) remain largely unknown, hindering the biological study of this modification. Here we report the identification of 2,580 bacterial lysine succinylation sites in 670 proteins and 2,803 lysine acetylation (Kac) sites in 782 proteins, representing the first lysine succinylation dataset and the largest Kac dataset in wild-type E. coli. We quantified dynamic changes of the lysine succinylation and Kac substrates in response to high glucose. Our data showed that high-glucose conditions led to more lysine-succinylated proteins and enhanced the abundance of succinyllysine peptides more significantly than Kac peptides, suggesting that glucose has a more profound effect on succinylation than on acetylation. We further identified CobB, a known Sir2-like bacterial lysine deacetylase, as the first prokaryotic desuccinylation enzyme. The identification of bacterial CobB as a bifunctional enzyme with lysine desuccinylation and deacetylation activities suggests that the eukaryotic Kac-regulatory enzymes may have enzymatic activities on various lysine acylations with very different structures. In addition, it is highly likely that lysine succinylation could have unique and more profound regulatory roles in cellular metabolism relative to lysine acetylation under some physiological conditions.Lysine acetylation (Kac)¹ is a dynamic and evolutionarily conserved post-translational modification (PTM) that is known to be involved in the regulation of diverse cellular processes (1–9). The status of this modification is controlled by two groups of enzymes with opposing enzymatic activities, lysine acetyltransferases that add an acetyl group to the lysine (Lys or K) residue, and histone lysine deacetylases (HDACs) that remove the acetyl group (10–16). HDACs are grouped into several categories (17): class I (HDAC1, -2, -3, and -8), class IIA (HDAC4, -5, -7, and -9), class IIB (HDAC6 and -10), class III (Sirt1–7), and class IV (HDAC11). The weak deacetylation activities of some HDACs (e.g. Sirt4–7 and HDAC4, -5, and -7–11), as well as the demonstration of Sirt5 as a desuccinylation and demalonylation enzyme, suggest that some HDAC enzymes have activities that are independent of acetylation (18, 19).For a long period of time, lysine acetylation was considered as a protein modification that was restricted to nuclei (20). The identification of cytosolic Kac substrates and the localization of some HDACs outside nuclei suggest a non-nuclear function of lysine acetylation (13, 21, 22). The first proteomic screening identified hundreds of substrate proteins in cytosolic and mitochondrial fractions and demonstrated high abundance of Kac in mitochondrial proteins and metabolic enzymes (23). This result implies that Kac has diverse non-nuclear roles and can regulate functions of metabolism and mitochondria (23). Since then, we and others have extensively characterized the cellular acetylome (5, 9, 24–26).The lysine succinylation (Ksucc) and lysine malonylation pathways are two PTM pathways that were recently identified and comprehensively validated in both bacterial and mammalian cells, with multiple substrate proteins identified, using HPLC-MS/MS, co-elution of synthetic peptides, isotopic labeling, Western blotting analysis using pan-anti-Ksucc antibodies, and proteomics analysis (18, 27). We also showed that Ksucc is present in core histones (29). In yeast histones, some Ksucc sites are located in regions where histones make close contact with DNA, suggesting that Ksucc sites may be involved in gene regulation by changing the chromatin structure (29). We then found that Sirt5, a member of the class III family of HDACs, can function as a desuccinylation enzyme in vitro and in vivo (18, 19). In a recent study, we revealed that Sirt5 is a key regulatory enzyme of Ksucc and that Ksucc proteins are abundant among a group of mitochondrial enzymes that are predominantly involved in fatty acid metabolism, amino acid degradation, and the tricarboxylic acid cycle (28). Importantly, Ksucc is very dynamic not only in mammalian cells, but also in bacteria (27, 29). These lines of evidence strongly suggest that lysine succinylation is likely an important PTM in the regulation of cellular functions.Although key elements of the Ksucc pathway are being identified in mammalian cells, their counterparts in bacteria remain largely unknown. We and others have used a proteomics approach to identify Kac substrates in bacteria (26, 30, 31, 52). The Sir2-like enzyme CobB is the best-studied protein deacetylase in bacteria (8). CobB was initially identified as an enzyme required for the activation of acetyl-CoA synthetase (8). Recently, CobB was shown to play roles in bacterial energy metabolism (31) and stress response (32). Those studies indicated that Kac is an evolutionarily conserved PTM with a role in energy metabolism in prokaryotes. Nevertheless, dynamic changes of lysine acetylation in bacteria have not been studied. In addition, substrates of lysine succinylation and their regulatory enzymes are not known.In this paper, we report a quantitative proteomic approach based on stable isotope labeling by amino acids in cell culture (SILAC) to identify and quantify changes in bacterial lysine succinylation, as well as lysine acetylation, in response to glucose, a major energy source. Our screening detected 2,580 lysine-succinylated sites in 670 proteins and 2,803 Kac sites in 782 proteins in Escherichia coli. Our quantitative proteomics data show that glucose had a more profound effect on Ksucc than on Kac. In addition, we found that CobB, a known prokaryotic deacetylase, had dual enzymatic activities to catalyze the removal of two structurally different lysine acyl groups, acetyl and succinyl, from the modified lysine residues.