The ubiquitously expressed family of ␣-actinins bridges actin filaments to stabilize adhesions, a process disrupted during growth factor-induced migration of cells. During the dissolution of the actin cytoskeleton, actinins are phosphorylated on tyrosines, although the consequences of this are unknown. We expressed the two isoforms of human ␣-actinin in murine fibroblasts that express human epidermal growth factor receptor (EGFR) and found that both ␣-actinin 1 (ACTN1) and ␣-actinin 4 (ACTN4) were phosphorylated on tyrosine residues after stimulation with EGF, although ACTN4 was phosphorylated to the greater extent. This required the activation of Src protein-tyrosine kinase and p38-MAPK (and phosphoinositide trisphosphate kinase in part) but not MEK/ERK or Rac1, as determined by inhibitors. The EGF-induced phosphorylation sites of ACTN4 were mapped to tyrosine 4, the major site, and tyrosine 31, the minor one. Truncation mutagenesis showed that the C-terminal domains of ACTN4 (amino acids 300 -911), which cross-link the actin binding head domains, act as an inhibitory domain for both actin binding and EGF-mediated phosphorylation. These two properties were mutually exclusive; removal of the C terminus enhanced actin binding of ACTN4 mutants while limiting EGF-induced phosphorylation, and conversely EGF-stimulated phosphorylation of ACTN4 decreased its affinity to actin. Interestingly, a phosphomimetic of tyrosine 265 (which can be found in carcinoma cells and lies near the K255E mutation that causes focal segmental glomerulosclerosis) demonstrated increased actin binding activity and susceptibility of ACTN4 to calpain-mediated cleavage; this variant also retarded cell spreading. Remarkably, either treatment of cells with low concentrations of latrunculin A, which has been shown to depolymerize F-actin, or the deletion of the actin binding domain (100 -252 amino acids) of ACTN4Y265E restored EGF-induced phosphorylation. An F-actin binding assay in vitro showed that Y4E/Y31E, a mimetic of diphosphorylated ACTN4, bound F-actin slightly compared with wild type (WT). Importantly, the EGF-mediated phosphorylation of ACTN4 at tyrosine 4 and 31 significantly inhibited multinucleation of proliferating NR6WT fibroblasts that overexpress ACTN4. These results suggest that EGF regulates the actin binding activity of ACTN4 by inducing tyrosyl-directed phosphorylation.Cell motility results from a complex and dynamic process that integrates adhesion and signaling receptors via intracellular signaling cascades at the level of the actin cytoskeleton. To accomplish locomotion, growth factor receptor occupancy leads to phosphorylation cascades that direct cytoskeletal liability and adhesion turnover. This requires actin filaments to be both unbridged and dissociated from the focal adhesions.
Calpain activity is required for de-adhesion of the cell body and rear to enable productive locomotion of adherent cells during wound repair and tumor invasion. Growth factors activate m-calpain (calpain 2, CAPN2) via ERK/mitogen-activated protein kinases, but only when these kinases are localized to the plasma membrane. We thus hypothesized that m-calpain is activated by epidermal growth factor (EGF) only when it is juxtaposed to the plasma membrane secondary to specific docking. Osmotic disruption of NR6 fibroblasts expressing the EGF receptor demonstrated m-calpain being complexed with the substratum-adherent membrane with this increasing in an EGF-dependent manner. m-Calpain colocalized with phosphoinositide biphosphate (PIP 2 ) with exogenous phospholipase C removal of phosphoinositides, specifically, PI(4,5)P 2 but not PI(4)P 1 or PIP 3 , releasing the bound m-calpain. Downregulation of phosphoinositide production by 1-butanol resulted in diminished PIP 2 in the plasma membrane and eliminated EGF-induced calpain activation. This PIP 2 -binding capacity resided in domain III of calpain, which presents a putative C2-like domain. This active conformation of this domain appears to be partially masked in the holoenzyme as both activation of m-calpain by phosphorylation at serine 50 and expression of constitutively active phosphorylation mimic glutamic acid-increased m-calpain binding to the membrane, consistent with blockade of this cascade diminishing membrane association. Importantly, we found that m-calpain was enriched toward the rear of locomoting cells, which was more pronounced in the plasma membrane footprints; EGF further enhanced this enrichment, in line with earlier reports of loss of PIP 2 in lamellipodia of motile cells. These data support a model of m-calpain binding to PIP 2 concurrent with and likely to enable ERK activation and provides a mechanism by which cell de-adhesion is directed to the cell body and tail as phospholipase C-␥ hydrolyzes PIP 2 in the protruding lamellipodia.Cell motility is a complex process involving a sequence of events consisting of extension of a lamellipodium, formation of new adhesions at the leading edge, contraction of the cell body, and detachment of the rear of the cell (35, 47). These separate events must work in a coordinated effort to provide persistent cell movement in one direction. Rear detachment has been shown to be a rate-limiting step during both haptokinetic (26, 45) and growth factor-induced chemokinetic (22, 32, 56) motility. This subcellular asymmetry of processes occurs even in the absence of an externally imposed gradient of cues (35, 62), suggesting an intracellular segregation of biochemical controls. While progress has been made in deciphering the signaling gradients during ameboid movement in yeast (28, 37), the situation in mammalian fibroblasts and epithelial cells is less clear (47).Extrinsic signals, including growth factors and the extracellular matrix, initiate intracellular signal cascades leading to biophysical changes in the cell (60,...
A major function of TFIID is core promoter recognition. TFIID consists of TATA-binding protein (TBP) and 14 TBP-associated factors (TAFs). Most of them contain a histone fold domain (HFD) that lacks the DNAcontacting residues of histones. Whether and how TAF HFDs contribute to core promoter DNA binding are yet unresolved. Here we examined the DNA binding activity of TAF9, TAF6, TAF4b, and TAF12, which are related to histones H3, H4, H2A, and H2B, respectively. Each of these TAFs has intrinsic DNA binding activity adjacent to or within the HFD. The DNA binding domains were mapped to evolutionarily conserved and essential regions. Remarkably, HFD-mediated interaction enhanced the DNA binding activity of each of the TAF6-TAF9 and TAF4b-TAF12 pairs and of a histone-like octamer complex composed of the four TAFs. Furthermore, HFD-mediated interaction stimulated sequence-specific binding by TAF6 and TAF9. These results suggest that TAF HFDs merge with other conserved domains for efficient and specific core promoter binding.Transcription of protein-encoding genes in eukaryotes involves the assembly of RNA polymerase II and general transcription factors (GTFs) on the core promoter to form a preinitiation complex (PIC). TFIID is the major DNA-binding GTF. It is composed of the TATA-binding protein (TBP) and 14 TBP-associated factors (TAFs). TAFs regulate transcription at multiple levels. Certain TAFs interact with activators to facilitate PIC formation (2, 18) and transcription reinitiation (1). TAFs also have a role in recognition and binding to core promoter elements. DNase I footprinting revealed direct contact of TAFs with sequences upstream and downstream of the TATA box (11,19,23,26,29,35). TAF1-TAF2 (TAF II 250-TAF II 150) binds the Initiator element (7,33,34), multiple TAFs were cross-linked to the adenovirus major late (AdML) promoter (24), and Drosophila melanogaster TAF6 (dTAF6) and dTAF9 (dTAF II 60-dTAF II 42) were cross-linked to the downstream promoter element (DPE) (5). Some TAFs are TFIID specific, but others are shared by other transcription regulatory complexes (3,4,15,22,25,28).One common feature found in 9 out of the 14 TAFs is the histone fold motif (for a review see reference 13). This motif has been established as an essential protein-protein interaction domain that facilitates assembly of TFIID in a manner analogous to that for histones (17,30). TAF6 and TAF9 are structurally related to histones H4 and H3, respectively (38). TAF12 is similar to H2B, TAF4 contains an H2A-like domain, and both interact with each other via the histone fold domain (HFD) (14,16,30). In vitro Saccharomyces cerevisiae TAF6 (yTAF6)-yTAF9 can assemble with yTAF12-yTAF4 to form a histone octamer-like structure (30).The nucleosome-like interaction of TFIID with DNA (24) and the presence of histone fold TAFs within this complex have led to the proposal that a nucleosome-like octamer within TFIID may be involved in direct DNA binding (17). However, the issue has remained elusive. First, there is no experimental evidence for D...
m-calpain plays a critical role in cell migration enabling rear de-adhesion of adherent cells by cleaving structural components of the adhesion plaques. Growth factors and chemokines regulate keratinocyte, fibroblast, and endothelial cell migration by modulating m-calpain activity. Growth factor receptors activate m-calpain secondary to phosphorylation on serine 50 by ERK. Concurrently, activated m-calpain is localized to its inner membrane milieu by binding to phosphatidylinositol 4,5-bisphosphate (PIP 2 ). Opposing this, CXCR3 ligands inhibit cell migration by blocking m-calpain activity secondary to a PKAmediated phosphorylation in the C2-like domain. The failure of m-calpain activation in the absence of PIP 2 points to a key regulatory role, although whether this PIP 2 -mediated membrane localization is regulatory for m-calpain activity or merely serves as a docking site for ERK phosphorylation is uncertain. Herein, we report the effects of two CXCR3 ligands, CXCL11/IP-9/I-TAC and CXCL10/IP-10, on the EGF-and VEGF-induced redistribution of m-calpain in human fibroblasts and endothelial cells. The two chemokines block the tail retraction and, thus, the migration within minutes, preventing and reverting growth factor-induced relocalization of m-calpain to the plasma membrane of the cells. PKA phosphorylation of m-calpain blocks the binding of the protease to PIP 2 . Unexpectedly, we found that this was due to membrane anchorage itself and not merely serine 50 phosphorylation, as the farnesylation-induced anchorage of m-calpain triggers a strong activation of this protease, leading notably to an increased cell death. Moreover, the ERK and PKA phosphorylations have no effect on this membrane-anchored m-calpain. However, the presence of PIP 2 is still required for the activation of the anchored m-calpain. In conclusion, we describe a novel mechanism of m-calpain activation by interaction with the plasma membrane and PIP 2 specifically, this phosphoinositide acting as a cofactor for the enzyme. The phosphorylation of m-calpain by ERK and PKA by growth factors and chemokines, respectively, act in cells to regulate the enzyme only indirectly by controlling its redistribution.Calpains are intracellular cysteine proteases involved in numerous physiological and pathological phenomena, such as embryo development and tumor invasion (1). Among the 15 members of the calpain family, the two ubiquitous calpains, calpain 1 and calpain 2, are the best described. They form with the calpain small subunit 1 (calpain S1) two heterodimers, -calpain for calpain 1 and m-calpain for calpain 2, that are strongly involved in the regulation of cell motility. Cell migration was previously described as a four-stage process: cell membrane protrusion, adhesion to the substrate, contraction of the cell body, and finally, release of the adhesion contacts at the rear of the cell (2-4). Cell migration is, thus, governed by a succession of adhesion and de-adhesion steps, and a balance between these two processes is required for an optimal cell mo...
Backgroundα-Actinins cross-link actin filaments, with this cross-linking activity regulating the formation of focal adhesions, intracellular tension, and cell migration. Most non-muscle cells such as fibroblasts express two isoforms, α-actinin-1 (ACTN1) and α-actinin-4 (ACTN4). The high homology between these two isoforms would suggest redundancy of their function, but recent studies have suggested different regulatory roles. Interestingly, ACTN4 is phosphorylated upon growth factor stimulation, and this loosens its interaction with actin.Methodology/Principal FindingsUsing molecular, biochemical and cellular techniques, we probed the cellular functions of ACTN4 in fibroblasts. Knockdown of ACTN4 expression in murine lung fibroblasts significantly impaired cell migration, spreading, adhesion, and proliferation. Surprisingly, knockdown of ACTN4 enhanced cellular compaction and contraction force, and increased cellular and nuclear cross-sectional area. These results, except the increased contractility, are consistent with a putative role of ACTN4 in cytokinesis. For the transcellular tension, knockdown of ACTN4 significantly increased the expression of myosin light chain 2, a element of the contractility machinery. Re-expression of wild type human ACTN4 in ACTN4 knockdown murine lung fibroblasts reverted cell spreading, cellular and nuclear cross-sectional area, and contractility back towards baseline, demonstrating that the defect was due to absence of ACTN4.SignificanceThese results suggest that ACTN4 is essential for maintaining normal spreading, motility, cellular and nuclear cross-sectional area, and contractility of murine lung fibroblasts by maintaining the balance between transcellular contractility and cell-substratum adhesion.
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