To become migratory, cells must reorganize their connections to the substratum, and during locomotion they must break rear attachments. The molecular and biochemical mechanisms underlying these biophysical processes are unknown. Recent studies have implicated both extracellular signal-regulated kinase/mitogen-activated protein (ERK/MAP) kinase and calpain (EC 3.4.22.17) in these processes, but it is uncertain whether these are two distinct pathways acting on different modes of motility. We report that cell deadhesion involved in epidermal growth factor (EGF) receptor-mediated fibroblast motility requires activation of M-calpain downstream of ERK/MAP kinase signaling. NR6 fibroblasts expressing full-length wild type epidermal growth factor receptor required both calpain and ERK activation, as demonstrated by pharmacological inhibitors (calpeptin and calpain inhibitor I and PD98059, respectively) for EGF-induced deadhesion and motility. EGF induced rapid activation of calpain that was preventable by molecular inhibition of the Ras-Raf-MEK but not phospholipase C␥ signaling pathway, and calpain was stimulated by transfection of constitutively active MEK. Enhanced calpain activity was not mirrored by increased calpain protein levels or decreased levels of its endogenous inhibitor calpastatin. The link between ERK/MAP kinase signaling and cell motility required the M-isoform of calpain (calpain II), as determined by specific antisense-mediated down-regulation. These data promote a previously undescribed signaling pathway of ERK/MAP kinases activating calpain to destabilize cell-substratum adhesions in response to EGF stimulation.Cell motility is a central process involved in many physiological events including tumor invasion, embryonic development, and wound healing. Cell motility has been found to be a very complex biophysical process involving multiple factors working together to accomplish concerted movement. To begin to understand the molecular bases of motility, fibroblast cell motility has been deconstructed into four separate events: extension of the lammelipod, formation of new focal adhesions at the leading edge, breaking of adhesions at the trailing edge, and translocation of the cell mass (1). Failure of any one of these steps is sufficient to prevent cell motility (2, 3).These motility events are regulated by both cell intrinsic and extrinsic properties (4, 5). Among the latter are regulatory inputs from growth factor receptors and the strength and nature of cell-substratum interactions. Previously, it had been demonstrated that motility of adherent cells occurs in a biphasic relationship to adhesiveness of the surface (6, 7). Recently, we have shown that this also holds for growth factor-induced motility, in that epidermal growth factor (EGF) 1 receptor-mediated fibroblast motility is noted only at intermediate levels of cell adhesiveness to substrata (8, 9). Thus, signals that regulate adhesiveness to the substratum are of high importance, since the rear deadhesion of the cell from its substrate can be ...

Biochemical and structural analysis of two features of kinase structure, the “R-spine” and “Shell,” afford a detailed insight into the regulation of eukaryotic protein kinases.

No abstract

The mechanism of PKAc-dependent NF-κB activation and subsequent translocation into the nucleus is not well defined. Previously, we showed that A kinase interacting protein 1 (AKIP1) was important for binding and retaining PKAc in the nucleus. Since then, other groups have demonstrated that AKIP1 binds the p65 subunit of NF-κB and regulates its transcriptional activity through the phosphorylation at Ser 276 by PKAc. However, little is known about the formation and activation of the PKAc/AKIP1/p65 complex and the rate at which it enters the nucleus. Initially, we found that the AKIP1 isoform (AKIP 1A) simultaneously binds PKAc and p65 in resting and serum starved cells. Using peptide arrays, we refined the region of AKIP 1A binding on PKAc and mapped the non-overlapping regions on AKIP 1A where PKAc and p65 bind. A peptide to the amino-terminus of PKAc (CAT 1-29) was generated to specifically disrupt the interaction between AKIP 1A and PKAc to study nuclear import of the complex. The rate of p65 nuclear translocation was monitored in the presence or absence of overexpressed AKIP 1A and/or (CAT 1-29). Enhanced nuclear translocation of p65 was observed in the presence of overexpressed AKIP1 and/or CAT 1-29 in cells stimulated with TNFα, and this correlated with decreased phosphorylation of serine 276. To determine whether PKAc phosphorylation of p65 in the cytosol regulated nuclear translocation, serine 276 was mutated to alanine or aspartic acid. Accelerated nuclear accumulation of p65 was observed in the alanine mutant, while the aspartic acid mutation displayed slowed nuclear translocation kinetics. In addition, enhanced nuclear translocation of p65 was observed when PKAc was knocked-down by siRNA. Taken together, these results suggest that AKIP 1A acts to scaffold PKAc to NF-κB in the cytosol by protecting the phosphorylation site and thereby regulating the rate of nuclear translocation of p65.

cAMP-dependent protein kinase (PKA) regulates a myriad of functions in the heart, including cardiac contractility, myocardial metabolism, and gene expression. However, a molecular integrator of the PKA response in the heart is unknown. Here, we show that the PKA adaptor A-kinase interacting protein 1 (AKIP1) is up-regulated in cardiac myocytes in response to oxidant stress. Mice with cardiac gene transfer of AKIP1 have enhanced protection to ischemic stress. We hypothesized that this adaptation to stress was mitochondrialdependent. AKIP1 interacted with the mitochondrial localized apoptosis inducing factor (AIF) under both normal and oxidant stress. When cardiac myocytes or whole hearts are exposed to oxidant and ischemic stress, levels of both AKIP1 and AIF were enhanced. AKIP1 is preferentially localized to interfibrillary mitochondria and up-regulated in this cardiac mitochondrial subpopulation on ischemic injury. Mitochondria isolated from AKIP1 genetransferred hearts showed increased mitochondrial localization of AKIP1, decreased reactive oxygen species generation, enhanced calcium tolerance, decreased mitochondrial cytochrome C release, and enhance phosphorylation of mitochondrial PKA substrates on ischemic stress. These observations highlight AKIP1 as a critical molecular regulator and a therapeutic control point for stress adaptation in the heart. ischemia/reperfusion | oxidative stress

During wound healing, dermal fibroblasts switch from a migratory, repopulating phenotype to a contractile, matrix-reassembling phenotype. The mechanisms controlling this switch are unknown. A possible explanation is suggested by the finding that chemokines that appear late in wound repair prevent growth factor-induced cell-substratum de-adhesion by blocking calpain activation. In this study, we tested the specific hypothesis that fibroblast contraction of the matrix is promoted by a pro-repair growth factor, epidermal growth factor, and is modulated by calpain-mediated release of adhesions. We employed an isometric force transduction system designed to measure the contraction of a collagen matrix under tension by a population of NR6 fibroblasts transfected with the human epidermal growth factor receptor. By maintaining a fixed level of strain, we could monitor both the initial contraction and subsequent relaxation of the matrix. Epidermal growth factor stimulated a transient, dose-dependent increase in matrix contraction that peaked within 60 minutes and then decayed over the ensuing 3 to 6 hours. Calpain inhibitor I (ALLN) prevented epidermal growth factor-stimulated cell de-adhesion and resulted in a significantly slower decay of matrix contraction, with only a slight decrease of the peak magnitude of contraction. The mitogen-activated protein kinase kinase-1-selective inhibitor PD 98059 that blocks signaling through the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway, required for epidermal growth factor receptor-mediated activation of calpain and de-adhesion, does not significantly affect the magnitude of matrix contraction within minutes of epidermal growth factor addition, but slows the decay similarly to calpain inhibition. Epidermal growth factor receptor signaling thus stimulates the complementary mechanisms of intracellular contractile force generation and calpain-mediated de-adhesion, which are known to coordinately facilitate cell migration. These findings suggest that calpain can act as a functional switch for transmission of intracellular contractile force to the surrounding matrix, with calpain-mediated de-adhesion reducing this transmission and corresponding matrix contraction. Countervailing processes that down-regulate calpain activation can, accordingly, direct the transition of cell function from locomotion to matrix contraction.

A current model of growth factor-induced cell motility invokes integration of diverse biophysical processes required for cell motility, including dynamic formation and disruption of cell/substratum attachments along with extension of membrane protrusions. To define how these biophysical events are actuated by biochemical signaling pathways, we investigate here whether epidermal growth factor (EGF) induces disruption of focal adhesions in fibroblasts. We find that EGF treatment of NR6 fibroblasts presenting full-length WT EGF receptors (EGFR) reduces the fraction of cells presenting focal adhesions from approximately 60% to approximately 30% within 10 minutes. The dose dependency of focal adhesion disassembly mirrors that for EGF-enhanced cell motility, being noted at 0.1 nM EGF. EGFR kinase activity is required as cells expressing two kinase-defective EGFR constructs retain their focal adhesions in the presence of EGF. The short-term (30 minutes) disassembly of focal adhesions is reflected in decreased adhesiveness of EGF-treated cells to substratum. We further examine here known motility-associated pathways to determine whether these contribute to EGF-induced effects. We have previously demonstrated that phospholipase C(gamma) (PLCgamma) activation and mobilization of gelsolin from a plasma membrane-bound state are required for EGFR-mediated cell motility. In contrast, we find here that short-term focal adhesion disassembly is induced by a signaling-restricted truncated EGFR (c'973) which fails to activate PLCgamma or mobilize gelsolin. The PLC inhibitor U73122 has no effect on this process, nor is the actin severing capacity of gelsolin required as EGF treatment reduces focal adhesions in gelsolin-devoid fibroblasts, further supporting the contention that focal adhesion disassembly is signaled by a pathway distinct from that involving PLCgamma. Because both WT and c'973 EGFR activate the erk MAP kinase pathway, we additionally explore here this signaling pathway, not previously associated with growth factor-induced cell motility. Levels of the MEK inhibitor PD98059 that block EGF-induced mitogenesis and MAP kinase phosphorylation also abrogate EGF-induced focal adhesion disassembly and cell motility. In summary, we characterize for the first time the ability of EGFR kinase activity to directly stimulate focal adhesion disassembly and cell/substratum detachment, in relation to its ability to stimulate migration. Furthermore, we propose a model of EGF-induced motogenic cell responses in which the PLCgamma pathway stimulating cell motility is distinct from the MAP kinase-dependent signaling pathway leading to disassembly and reorganization of cell-substratum adhesion.

Ischemic preconditioning, an endogenous protective mechanism to salvage ischemic myocardium, was described nearly 25 years ago; however, there has been little to no clinical translation. Further, there has been a critical lack of knowledge to understand the molecular mechanisms that bring about the protection. Nuclear factor‐kappa B (NF‐κB) and protein kinase A (PKA) are key regulators of gene transcription in the heart and have contradictory roles in cell death and survival. Activation of NF‐κB and PKA has been shown to be protective as well as promote cardiac cell death in the setting of ischemia or oxidant stress. The answer as to why these two important regulators of gene transcription and cardiac physiology produce such a dichotomous response upon stress induction may provide insights into how protective signaling in the heart is integrated. We have discovered a novel scaffolding protein known as A‐kinase interacting protein 1 (AKIP1) that is expressed at low levels in the heart and is induced by stress. Our data shows that AKIP1 binds to and regulates nuclear localization of PKA catalytic subunit (PKAc) and increases nuclear PKAc activity. Further, AKIP1 interacts with and enhances NF‐κB nuclear localization in a PKAc dependent manner where disruption of AKIP1 binding to PKAc enhances nuclear NF‐κB. In cardiomyocytes and in the heart, oxidant/ischemic stress caused an increase of AKIP1 levels. Further, our data suggests that overexpression of AKIP1 was protective to the heart. Therefore, we infer that AKIP1 may be a key molecular regulator/scaffold that assembles PKAc and NF‐κB signaling complexes to alter the physiologic response of the heart in the basal and stressed states. Understanding the dynamics and physiologic implications of the interaction of PKAc and NF‐κB with AKIP1 may provide a novel therapeutic control point for limiting cardiac injury associated with ischemic stress.