Phosphorylation of Thr-516 and Ser-520 in the Kinase Activation Loop of MEKK3 Is Required for Lysophosphatidic Acid-mediated Optimal I��B Kinase �� (IKK��)/Nuclear Factor-��B (NF-��B) Activation

MEKK3 serves as a critical intermediate signaling molecule in lysophosphatidic acid-mediated nuclear factor-κB (NF-κB) activation. However, the precise regulation for MEKK3 activation at the molecular level is still not fully understood. Here we report the identification of two regulatory phosphorylation sites at Thr-516 and Ser-520 within the kinase activation loop that is essential for MEKK3-mediated IκB kinase β (IKKβ)/NF-κB activation. Substitution of these two residues with alanine abolished the ability of MEKK3 to activate IKKβ/NF-κB, whereas replacement with acidic residues rendered MEKK3 constitutively active. Furthermore, substitution of these two residues with alanine abolished the ability of MEKK3 to mediate lysophosphatidic acid-induced optimal IKKβ/NF-κB activation.     

Molecular Basis for Class V ��-Tubulin Effects on Microtubule Assembly and Paclitaxel Resistance

Vertebrates produce at least seven distinct β-tubulin isotypes that coassemble into all cellular microtubules. The functional differences among these tubulin isoforms are largely unknown, but recent studies indicate that tubulin composition can affect microtubule properties and cellular microtubule-dependent behavior. One of the isotypes whose incorporation causes the largest change in microtubule assembly is β5-tubulin. Overexpression of this isotype can almost completely destroy the microtubule network, yet it appears to be required in smaller amounts for normal mitotic progression. Moderate levels of overexpression can also confer paclitaxel resistance. Experiments using chimeric constructs and site-directed mutagenesis now indicate that the hypervariable C-terminal region of β5 plays no role in these phenotypes. Instead, we demonstrate that two residues found in β5 (Ser-239 and Ser-365) are each sufficient to inhibit microtubule assembly and confer paclitaxel resistance when introduced into β1-tubulin; yet the single mutation of residue Ser-239 in β5 eliminates its ability to confer these phenotypes. Despite the high degree of conservation among β-tubulin isotypes, mutations affecting residue 365 demonstrate that amino acid substitutions can be context sensitive; i.e. an amino acid change in one isotype will not necessarily produce the same phenotype when introduced into a different isotype. Modeling studies indicate that residue Cys-239 of β1-tubulin is close to a highly conserved Cys-354 residue suggesting the possibility that disulfide formation could play a significant role in the stability of microtubules formed with β1- but not with β5-tubulin.Microtubules are needed to organize the Golgi apparatus and endoplasmic reticulum, maintain cell shape, construct ciliary and flagellar axonemes, and ensure the accurate segregation of genetic material prior to cell division. These cytoskeletal structures assemble from α- and β-tubulin heterodimers to form long cylindrical filaments that exist in a state of dynamic equilibrium characterized by stochastic episodes of slow growth and rapid shrinkage (1). Impairment of normal dynamic behavior has serious consequences for cell proliferation and thus makes microtubules an attractive target for drug development (2).Vertebrates express multiple β-tubulin genes that produce highly homologous proteins differing most notably in their C-terminal 15–20 amino acids (3, 4). These variable C-terminal sequences are conserved across vertebrate species and have been used to classify β-tubulin genes into distinct isotypes (5). In mammals, for example, there are seven known isotypes designated by the numbers I, II, III, IVa, IVb, V, and VI. The functional significance of the C-terminal sequences is uncertain, but some studies suggest that they may be involved in binding or modulating the action of microtubule-interacting proteins (6–14). Additional amino acid differences are scattered throughout the primary sequence, but the functional role of these differences, if any, has not been elucidated. Although some β-tubulin isotypes are expressed in a tissue-specific manner (3), evidence indicates that microtubules incorporate all available isotypes, including transfected isotypes that are not normally produced in those cells (5, 15–17). Genetic experiments designed to test potential functional differences among the various β-tubulin isotypes have only demonstrated isotype-specific effects on the assembly of specialized microtubule-containing structures such as flagellar axonemes in Drosophila or 15-protofilament microtubules in Caenorhabditis elegans (18, 19). Thus, the consequences, if any, of producing multiple β-tubulin isoforms in vertebrate organisms remain elusive.Our recent work showed that conditional overexpression of isotypes β1, β2, and β4b has no effect on microtubule assembly or drug sensitivity in transfected Chinese hamster ovary (CHO)² cells (20). Similarly, expression of neuronal-specific β4a produced very minor effects on microtubule assembly but was able to increase sensitivity to paclitaxel, most likely through increased binding of the drug (21). On the other hand, high expression of neuronal-specific β3 reduced microtubule assembly, conferred low level resistance to paclitaxel, and inhibited cell growth (22). The most dramatic effects, however, were seen in cells transfected with β5, a minor but widely expressed isotype (23). Even modest overexpression of this isotype reduced microtubule assembly and conferred paclitaxel resistance, whereas high levels of expression (∼50% of total tubulin) caused fragmentation and a near complete loss of the microtubule cytoskeleton (24). Despite the toxicity associated with β5 overexpression, this isotype was recently shown to be required for normal mitotic progression and cell proliferation (25).Because of its importance for cell division, and the extreme phenotype associated with its overexpression, we sought to identify the structural differences between β5-tubulin and its more “normal” homolog, β1. Although there are 40 amino acid differences between the 2 isotypes, we report that most of the unique properties of β5 can be attributed to the presence of serine in place of cysteine at residue 239. This residue faces the colchicine binding pocket and is very close to a highly conserved Cys-354 residue. We propose that Ser-239 found in β5-tubulin may prevent formation of a disulfide bond that normally stabilizes microtubules.     

Inflammation Anergy in Human Intestinal Macrophages Is Due to Smad-induced I��B�� Expression and NF-��B Inactivation

Human intestinal macrophages contribute to tissue homeostasis in noninflamed mucosa through profound down-regulation of pro-inflammatory cytokine release. Here, we show that this down-regulation extends to Toll-like receptor (TLR)-induced cytokine release, as intestinal macrophages expressed TLR3–TLR9 but did not release cytokines in response to TLR-specific ligands. Likely contributing to this unique functional profile, intestinal macrophages expressed markedly down-regulated adapter proteins MyD88 and Toll interleukin receptor 1 domain-containing adapter-inducing interferon β, which together mediate all TLR MyD88-dependent and -independent NF-κB signaling, did not phosphorylate NF-κB p65 or Smad-induced IκBα, and did not translocate NF-κB into the nucleus. Importantly, transforming growth factor-β released from intestinal extracellular matrix (stroma) induced identical down-regulation in the NF-κB signaling and function of blood monocytes, the exclusive source of intestinal macrophages. Our findings implicate stromal transforming growth factor-β-induced dysregulation of NF-κB proteins and Smad signaling in the differentiation of pro-inflammatory blood monocytes into noninflammatory intestinal macrophages.     

N-Glycosylation of the I-like Domain of ��1 Integrin Is Essential for ��1 Integrin Expression and Biological Function: IDENTIFICATION OF THE MINIMAL N-GLYCOSYLATION REQUIREMENT FOR ��5��1*

N-Glycosylation of integrin α5β1 plays a crucial role in cell spreading, cell migration, ligand binding, and dimer formation, but the detailed mechanisms by which N-glycosylation mediates these functions remain unclear. In a previous study, we showed that three potential N-glycosylation sites (α5S3–5) on the β-propeller of the α5 subunit are essential to the functional expression of the subunit. In particular, site 5 (α5S5) is the most important for its expression on the cell surface. In this study, the function of the N-glycans on the integrin β1 subunit was investigated using sequential site-directed mutagenesis to remove the combined putative N-glycosylation sites. Removal of the N-glycosylation sites on the I-like domain of the β1 subunit (i.e. the Δ4-6 mutant) decreased both the level of expression and heterodimeric formation, resulting in inhibition of cell spreading. Interestingly, cell spreading was observed only when the β1 subunit possessed these three N-glycosylation sites (i.e. the S4-6 mutant). Furthermore, the S4-6 mutant could form heterodimers with either α5S3-5 or α5S5 mutant of the α5 subunit. Taken together, the results of the present study reveal for the first time that N-glycosylation of the I-like domain of the β1 subunit is essential to both the heterodimer formation and biological function of the subunit. Moreover, because the α5S3-5/β1S4-6 mutant represents the minimal N-glycosylation required for functional expression of the β1 subunit, it might also be useful for the study of molecular structures.Integrin is a heterodimeric glycoprotein that consists of both an α and a β subunit (1). The interaction between integrin and the extracellular matrix is essential to both physiologic and pathologic events, such as cell migration, development, cell viability, immune homeostasis, and tumorigenesis (2, 3). Among the integrin superfamily, β1 integrin can combine with 12 distinct α subunits (α1–11, αv) to form heterodimers, thereby acquiring a wide variety of ligand specificity (1, 4). Integrins are thought to be regulated by inside-out signaling mechanisms that provoke conformational changes, which modulate the affinity of integrin for the ligand (5). However, an increasing body of evidence suggests that cell-surface carbohydrates mediate a variety of interactions between integrin and its extracellular environment, thereby affecting integrin activity and possibly tumor metastasis as well (6–8).Guo et al. (9) reported that an increase in β1–6-GlcNAc sugar chains on the integrin β1 subunit stimulated cell migration. In addition, elevated sialylation of the β1 subunit, because of Ras-induced STGal-I transferase activity, also induced cell migration (10, 11). Conversely, cell migration and spreading were reduced by the addition of a bisecting GlcNAc, which is a product of N-acetylglucosaminyltransferase III (GnT-III),² to the α5β1 and α3β1 integrins (12, 13). Alterations of N-glycans on integrins might also regulate their cis interactions with membrane-associated proteins, including the epidermal growth factor receptor, the galectin family, and the tetraspanin family of proteins (14–19).In addition to the positive and negative regulatory effects of N-glycan, several research groups have reported that N-glycans must be present on integrin α5β1 for the αβ heterodimer formation and proper integrin-matrix interactions. Consistent with this hypothesis, in the presence of the glycosylation inhibitor, tunicamycin, normal integrin-substrate binding and transport to the cell surface are inhibited (20). Moreover, treatment of purified integrin with N-glycosidase F blocked both the inherent association of the subunits and the interaction between integrin and fibronectin (FN) (21). These results suggest that N-glycosylation is essential to the functional expression of α5β1. However, because integrin α5β1 contains 26 potential N-linked glycosylation sites, 14 in the α subunit and 12 in the β subunit, identification of the sites that are essential to its biological functions is key to understanding the molecular mechanisms by which N-glycans alter integrin function. Recently, our group determined that N-glycosylation of the β-propeller domain on the α5 subunit is essential to both heterodimerization and biological functions of the subunit. Furthermore, we determined that sites 3–5 are the most important sites for α5 subunit-mediated cell spreading and migration on FN (22). The purpose of this study was to clarify the roles of N-glycosylation of the β1 subunit. Therefore, we performed combined substitutions in the putative N-glycosylation sites by replacement of asparagine residues with glutamine residues. We subsequently introduced these mutated genes into β1-deficient epithelial cells (GE11). The results of these mutation experiments revealed that the N-glycosylation sites on the I-like domain of the β1 subunit, sites number 4–6 (S4-6), are essential to both heterodimer formation and biological functions, such as cell spreading.