P38 delta MAPK promotes breast cancer progression and lung metastasis by enhancing cell proliferation and cell detachment.

The protein p38 mitogen-activated protein kinase (MAPK) delta isoform (p38δ) is a poorly studied member of the MAPK family. Data analysis from The Cancer Genome Atlas database revealed that p38δ is highly expressed in all types of human breast cancers. Using a human breast cancer tissue array, we confirmed elevation in cancer tissue. The breast cancer mouse model, MMTV-PyMT (PyMT), developed breast tumors with lung metastasis; however, mice deleted in p38δ (PyMT/p38δ-/-) exhibited delayed primary tumor formation and highly reduced lung metastatic burden. At the cellular level, we demonstrate that targeting of p38δ in breast cancer cells, MCF-7 and MDA-MB-231 resulted in a reduced rate of cell proliferation. In addition, cells lacking p38δ also displayed an increased cell-matrix adhesion and reduced cell detachment. This effect on cell adhesion was molecularly supported by the regulation of the focal adhesion kinase by p38δ in the human breast cell lines. These studies define a previously unappreciated role for p38δ in breast cancer development and evolution by regulating tumor growth and altering metastatic properties. This study proposes MAPK p38δ protein as a key factor in breast cancer. Lack of p38δ resulted in reduced primary tumor size and blocked the metastatic potential to the lungs.

Mitogen and stress-activated kinases 1/2 regulate ischemia-induced hippocampal progenitor cell proliferation and neurogenesis.

Pathophysiological conditions such as cerebral ischemia trigger the production of new neurons from the neurogenic niche within the subgranular zone (SGZ) of the dentate gyrus. The functional significance of ischemia-induced neurogenesis is believed to be the regeneration of lost cells, thus contributing to post-ischemia recovery. However, the cell signaling mechanisms by which this process is regulated are still under investigation. Here, we investigated the role of mitogen and stress-activated protein kinases (MSK1/2) in the regulation of progenitor cell proliferation and neurogenesis after cerebral ischemia. Using the endothelin-1 model of ischemia, wild-type (WT) and MSK1(-/-)/MSK2(-/-) (MSK dKO) mice were injected with BrdU and sacrificed 2 days, 4 weeks, or 6 weeks later for the analysis of progenitor cell proliferation, neurogenesis, and neuronal morphology, respectively. We report a decrease in SGZ progenitor cell proliferation in MSK dKO mice compared to WT mice. Moreover, MSK dKO mice exhibited reduced neurogenesis and a delayed maturation of ischemia-induced newborn neurons. Further, structural analysis of neuronal arborization revealed reduced branching complexity in MSK dKO compared to WT mice. Taken together, this dataset suggests that MSK1/2 plays a significant role in the regulation of ischemia-induced progenitor cell proliferation and neurogenesis. Ultimately, revealing the cell signaling mechanisms that promote neuronal recovery will lead to novel pharmacological approaches for the treatment of neurodegenerative diseases such as cerebral ischemia.

Stress induced gene expression: a direct role for MAPKAP kinases in transcriptional activation of immediate early genes.

Immediate early gene (IEG) expression is coordinated by multiple MAP kinase signaling pathways in a signal specific manner. Stress-activated p38α MAP kinase is implicated in transcriptional regulation of IEGs via MSK-mediated CREB phosphorylation. The protein kinases downstream to p38, MAPKAP kinase (MK) 2 and MK3 have been identified to regulate gene expression at the posttranscriptional levels of mRNA stability and translation. Here, we analyzed stress-induced IEG expression in MK2/3-deficient cells. Ablation of MKs causes a decrease of p38α level and p38-dependent IEG expression. Unexpectedly, restoration of p38α does not rescue the full-range IEG response. Instead, the catalytic activity of MKs is necessary for the major transcriptional activation of IEGs. By transcriptomics, we identified MK2-regulated genes and recognized the serum response element (SRE) as a common promoter element. We show that stress-induced phosphorylation of serum response factor (SRF) at serine residue 103 is significantly reduced and that induction of SRE-dependent reporter activity is impaired and can only be rescued by catalytically active MK2 in MK2/3-deficient cells. Hence, a new function of MKs in transcriptional activation of IEGs via the p38α-MK2/3-SRF-axis is proposed which probably cooperates with MKs' role in posttranscriptional gene expression in inflammation and stress response.

Effects of anionic surfactant and salinity on the bioenergetics of juveniles of Centropomus parallelus (Poey).

The acute and sublethal chronic effects of sodium dodecyl sulphate (SDS) on the survival, metabolism, and growth of juveniles of Centropomus parallelus were investigated at three different salinities. Results of 96 h LC50 test showed that juveniles of C. parallelus were very sensitive to SDS in comparison to other species investigated. For each group of exposure to nominal concentrations of SDS (0.10 and 0.25 mg/L) and the control group (0.0 mg/L), at the different salinities (5, 20, and 30) there were significant differences in the specific growth rate, oxygen consumption, and ammonia excretion rates, O:N atomic ratio at the different exposure periods (15 and 30 days). There were also interactions between factors for the parameters investigated. The present results show a pronounced effect of SDS, mainly at the highest concentration and salinity, as well as after a long time of exposure.

Post-translational control of Nur77.

Nur77 is a nuclear orphan receptor that has been implicated in both cell survival and apoptosis. With the exception of T-cells, translocation of Nur77 to the cytoplasm promotes cell death, while its retention in the nucleus promotes survival and proliferation. Nur77 appears to be a true orphan receptor, indicating that its activity must be controlled by ligand-independent mechanisms. Here, we discuss the role of phosphorylation in the regulation of Nur77.

A second binding site revealed by C-terminal truncation of calpain small subunit, a penta-EF-hand protein.

The subunits in calpain and in the related penta-EF-hand (PEF) proteins are bound through contacts between the unpaired EF-hand 5 from each subunit. To study subunit binding further, a tetra-EF-hand 18 kDa N- and C-terminally truncated form of the calpain small subunit was prepared (18k). This protein does not combine with the calpain large subunit to form active calpain, but forms homodimers in solution, as shown by ultracentrifugation. The X-ray structure of the 18k protein in the presence of cadmium was solved to a resolution of 2.0 A. The structure of the monomer is almost identical to the known structure of the calpain small subunit, but the 18k protein forms an oligomer in the crystal by the use of two binding sites. One of these sites is an artefact arising from the C-terminal truncation, but the other is a naturally occurring site that is fully exposed to water in intact purified calpain. The characteristics of this site suggest that it may be important in binding other protein modulators involved in the regulation of calpain and of PEF proteins.

Reduced cell migration and disruption of the actin cytoskeleton in calpain-deficient embryonic fibroblasts.

The physiological functions and substrates of the calcium-dependent protease calpain remain only partly understood. The mu- and m-calpains consist of a mu- or m-80-kDa large subunit (genes Capn1 and Capn2), and a common 28-kDa small subunit (Capn4). To assess the role of calpain in migration, we used fibroblasts obtained from Capn4(-/-) mouse embryos. The cells lacked calpain activity on casein zymography and did not generate the characteristic calpain-generated spectrin breakdown product that is observed in wild-type cells. Capn4(-/-) cells had decreased migration rates and abnormal organization of the actin cytoskeleton with a loss of central stress fibers. Interestingly, these cells extended numerous thin projections and displayed delayed retraction of membrane protrusions and filopodia. The number of focal adhesions was decreased in Capn4(-/-) cells, but the cells had prominent vinculin-containing focal complexes at the cell periphery. The levels of the focal adhesion proteins, alpha-actinin, focal adhesion kinase (FAK), spectrin, talin, and vinculin, were the same in Capn4(+/+) and Capn4(-/-) cells. FAK, alpha-actinin, and vinculin were not cleaved in either cell type plated on fibronectin. However, proteolysis of the focal complex component, talin, was detected in the wild-type cells but not in the Capn4(-/-) cells, suggesting that calpain cleavage of talin is important during cell migration. Moreover, talin cleavage was again observed when calpain activity was partially restored in Capn4(-/-) embryonic fibroblasts by stable transfection with a vector expressing the rat 28-kDa calpain small subunit. The results demonstrate unequivocally that calpain is a critical regulator of cell migration and of the organization of the actin cytoskeleton and focal adhesions.

Phosphorylation of the protein kinase mutated in Peutz-Jeghers cancer syndrome, LKB1/STK11, at Ser431 by p90(RSK) and cAMP-dependent protein kinase, but not its farnesylation at Cys(433), is essential for LKB1 to suppress cell vrowth.

Peutz-Jeghers syndrome is an inherited cancer syndrome that results in a greatly increased risk of developing tumors in those affected. The causative gene is a protein kinase termed LKB1, predicted to function as a tumor suppressor. The mechanism by which LKB1 is regulated in cells is not known. Here, we demonstrate that stimulation of Rat-2 or embryonic stem cells with activators of ERK1/2 or of cAMP-dependent protein kinase induced phosphorylation of endogenously expressed LKB1 at Ser(431). We present pharmacological and genetic evidence that p90(RSK) mediated this phosphorylation in response to agonists that activate ERK1/2 and that cAMP-dependent protein kinase mediated this phosphorylation in response to agonists that activate adenylate cyclase. Ser(431) of LKB1 lies adjacent to a putative prenylation motif, and we demonstrate that full-length LKB1 expressed in 293 cells was prenylated by addition of a farnesyl group to Cys(433). Our data suggest that phosphorylation of LKB1 at Ser(431) does not affect farnesylation and that farnesylation does not affect phosphorylation at Ser(431). Phosphorylation of LKB1 at Ser(431) did not alter the activity of LKB1 to phosphorylate itself or the tumor suppressor protein p53 or alter the amount of LKB1 associated with cell membranes. The reintroduction of wild-type LKB1 into a cancer cell line that lacks LKB1 suppressed growth, but mutants of LKB1 in which Ser(431) was mutated to Ala to prevent phosphorylation of LKB1 were ineffective in inhibiting growth. In contrast, a mutant of LKB1 that cannot be prenylated was still able to suppress the growth of cells.

MSK1 is required for CREB phosphorylation in response to mitogens in mouse embryonic stem cells.

Mouse embryonic stem (ES) cells homozygous for disruption of the MSK1 gene had no detectable MSK1 activity. However, their activators (extracellular signal related kinase (ERK)1/ERK2) were stimulated normally in mitogen- and stress-activated protein kinase (MSK)1-/- and wild type cells in response to tetradecanoylphorbol acetate (TPA) and epidermal growth factor (EGF). TPA and EGF induced the phosphorylation of cyclic AMP-responsive element binding protein (CREB) at Ser-133 and ATF1 at Ser-63 in wild type cells and this was abolished by inhibition of the mitogen-activated protein kinase cascade. In contrast, the TPA- and EGF-induced phosphorylation of CREB/ATF1 was barely detectable in MSK1-/- cells. However, basal and forskolin-induced phosphorylation was similar, indicating that the MSK1 'knockout' did not prevent CREB phosphorylation by cyclic AMP-dependent protein kinase. Thus MSK1 is required for CREB and ATF1 phosphorylation after mitogenic stimulation of ES cells.

Roles of individual EF-hands in the activation of m-calpain by calcium.

m-Calpain is a heterodimeric, cytosolic, thiol protease, which is activated by Ca(2+)-binding to EF-hands in the C-terminal domains of both subunits. There are four potential Ca(2+)-binding EF-hands in each subunit, but their relative affinities for Ca(2+) are not known. In the present study mutations were made in both subunits to reduce the Ca(2+)-binding affinity at one or more EF-hands in one or both subunits. X-ray crystallography of some of the mutated small subunits showed that Ca(2+) did not bind to the mutated EF-hands, but that its binding at other sites was not affected. The structures of the mutant small subunits in the presence of Ca(2+) were otherwise identical to that of the Ca(2+)-bound wild-type small subunit. In the whole enzyme the wild-type macroscopic Ca(2+) requirement (K(d)) was approx. 350 microM. The mutations did not affect the maximum specific activity of the enzyme, but caused increases in K(d), which were characteristic of each site. All the EF-hands could be mutated in various combinations without loss of activity, but preservation of at least one wild-type EF-hand 3 sequence was required to maintain K(d) values lower than 1 mM. The results suggest that all the EF-hands can contribute co-operatively to calpain activation, but that EF-hand 3, in both subunits, has the highest intrinsic affinity for Ca(2+) and provides the major driving force for conformational change.

Disruption of the murine calpain small subunit gene, Capn4: calpain is essential for embryonic development but not for cell growth and division.

Calpains are a family of Ca(2+)-dependent intracellular cysteine proteases, including the ubiquitously expressed micro- and m-calpains. Both mu- and m-calpains are heterodimers, consisting of a distinct large 80-kDa catalytic subunit, encoded by the genes Capn1 and Capn2, and a common small 28-kDa regulatory subunit (Capn4). The physiological roles and possible functional distinctions of mu- and m-calpains remain unclear, but suggested functions include participation in cell division and migration, integrin-mediated signal transduction, apoptosis, and regulation of cellular control proteins such as cyclin D1 and p53. Homozygous disruption of murine Capn4 eliminated both mu- and m-calpain activities, but this did not affect survival and proliferation of cultured embryonic stem cells or embryonic fibroblasts, or the early stages of organogenesis. However, mutant embryos died at midgestation and displayed defects in the cardiovascular system, hemorrhaging, and accumulation of erythroid progenitors.

The role of 3-phosphoinositide-dependent protein kinase 1 in activating AGC kinases defined in embryonic stem cells.

BACKGROUND: Protein kinase B (PKB), and the p70 and p90 ribosomal S6 kinases (p70 S6 kinase and p90 Rsk, respectively), are activated by phosphorylation of two residues, one in the 'T-loop' of the kinase domain and, the other, in the hydrophobic motif carboxy terminal to the kinase domain. The 3-phosphoinositide-dependent protein kinase 1 (PDK1) activates many AGC kinases in vitro by phosphorylating the T-loop residue, but whether PDK1 also phosphorylates the hydrophobic motif and whether all other AGC kinases are substrates for PDK1 is unknown. RESULTS: Mouse embryonic stem (ES) cells in which both copies of the PDK1 gene were disrupted were viable. In PDK1(-/-) ES cells, PKB, p70 S6 kinase and p90 Rsk were not activated by stimuli that induced strong activation in PDK1(+/+) cells. Other AGC kinases - namely, protein kinase A (PKA), the mitogen- and stress-activated protein kinase 1 (MSK1) and the AMP-activated protein kinase (AMPK) - had normal activity or were activated normally in PDK1(-/-) cells. The insulin-like growth factor 1 (IGF1) induced PKB phosphorylation at its hydrophobic motif, but not at its T-loop residue, in PDK1(-/-) cells. IGF1 did not induce phosphorylation of p70 S6 kinase at its hydrophobic motif in PDK1(-/-) cells. CONCLUSIONS: PDK1 mediates activation of PKB, p70 S6 kinase and p90 Rsk in vivo, but is not rate-limiting for activation of PKA, MSK1 and AMPK. Another kinase phosphorylates PKB at its hydrophobic motif in PDK1(-/-) cells. PDK1 phosphorylates the hydrophobic motif of p70 S6 kinase either directly or by activation of another kinase.

Crystallization and X-ray crystallographic analysis of m-calpain, a Ca2+-dependent protease.

The absolute requirement of Ca(2+) for proteolytic activity is a feature unique to the calpains, a family of heterodimeric cysteine proteases. Conditions are described which give rise to diffraction-quality crystals of m-calpain in two crystal forms, P1 and P2(1). Data have been collected from native crystals of m-calpain in both P1 and P2(1) forms, to 2.6 and 2.15 A, respectively. Selenomethionine-containing crystals have been grown in both forms, and anomalous data from the P2(1) selenomethionine enzyme provided the location of 17 of the 19 Se atoms in the protein.

m-Calpain subunits remain associated in the presence of calcium.

The hypothesis that calpain subunits dissociate in the presence of Ca2+ has been tested by methods which avoid interference by Ca2+-induced aggregation and large subunit autolysis. Inactive Cys105Ser-m-calpain, bound either to Ni-NTA-agarose or to immobilized casein, after incubation with Ca2+, could be recovered in high yield as a heterodimer. Natural bovine m-calpain, after irreversible inhibition with Z-LLY-CHN2, also bound to immobilized casein and was eluted as a heterodimer. The Ca2+ requirements of calpain containing a small subunit with EF-hand mutations were higher, both before and after autolysis, than those of wild-type calpain. In mixtures of wild-type and mutant enzymes, subunit exchange did not occur in the presence of Ca2+. The results demonstrate that the subunits in both natural and recombinant m-calpain, in the given experimental conditions, remain associated in the presence of Ca2+ both before and after autolysis.

Structure of the mouse calpain small subunit gene.

The calpains comprise a family of heterodimeric (80+28 kDa) Ca2+-dependent cysteine proteases, probably having roles in signal transduction and cytoskeletal remodelling. We describe cloning and sequencing of the 28 kDa calpain subunit cDNA from mouse (coding for 268 amino acids), and characterization of its gene. The gene spans 7 kb and contains 11 exons. The promoter region, like those of other calpain genes, lacks an obvious TATA box, but contains several Sp1 binding sites.

The effects of truncations of the small subunit on m-calpain activity and heterodimer formation.

In order to study subunit interactions in calpain, the effects of small subunit truncations on m-calpain activity and heterodimer formation have been measured. It has been shown previously that active calpain is formed by co-expression of the large subunit (80 kDa) of rat m-calpain with a delta 86 form (21 kDa) of the small subunit. cDNA for the full-length 270 amino acid (28.5 kDa) rat calpain small subunit has now been cloned, both with and without an N-terminal histidine tag (NHis10). The full-length small subunit constructs yielded active calpains on co-expression with the large subunit, and the small subunit was autolysed to 20 kDa on exposure of these calpains to Ca2+. A series of deletion mutants of the small subunit, NHis10-delta 86, -delta 99, -delta 107, and -delta 116, gave active heterodimeric calpains with unchanged specific activities, although in decreasing yield, and with a progressive decrease in stability. NHis10-delta 125 formed a heterodimer which was inactive and unstable. Removal of 25 C-terminal residues from delta 86, leaving residues 87-245, abolished both activity and heterodimer formation. The results show that: (a) generation of active m-calpain in Escherichia coli requires heterodimer formation; (b) small subunit residues between 94 and 116 contribute to the stability of the active heterodimer but do not directly affect the catalytic mechanism; (c) residues in the region 245-270 are essential for subunit binding. Finally, it was shown that an inactive mutant Cys103-->Ser-80k/delta 86 calpain, used in order to preclude autolysis, did not dissociate in the presence of Ca2+, a result which does not support the proposal that Ca(2+)-induced dissociation is involved in calpain activation.

Structure of a calpain Ca(2+)-binding domain reveals a novel EF-hand and Ca(2+)-induced conformational changes.

The crystal structure of a Ca(2+)-binding domain (dVI) of rat m-calpain has been determined at 2.3 A resolution, both with and without bound Ca2+. The structures reveal a unique fold incorporating five EF-hand motifs per monomer, three of which bind calcium at physiological calcium concentrations, with one showing a novel EF-hand coordination pattern. This investigation gives us a first view of the calcium-induced conformational changes, and consequently an insight into the mechanism of calcium induced activation in calpain. The crystal structures reveal a dVI homodimer which provides a preliminary model for the subunit dimerization in calpain.

Autolysis, Ca2+ requirement, and heterodimer stability in m-calpain.

The roles of N-terminal autolysis of the large (80 kDa) and small (28 kDa) subunits in activation of rat m-calpain, in lowering its Ca2+ requirement, and in reducing its stability have been investigated with heterodimeric recombinant calpains containing modified subunits. Both autolysis and [Ca2+]0.5 were influenced by the ionic strength of the buffers, which accounts for the wide variations in previous reports. Autolysis of the small subunit (from 28 to 20 kDa) was complete within 1 min but did not alter either the Ca2+ requirement ([Ca2+]0.5) or the stability of the enzyme. Autolysis of the NHis10-80k large subunit at Ala9-Lys10 is visible on gels, was complete within 1 min, and caused a drop in [Ca2+]0.5 from 364 to 187 microM. The lower value of [Ca2+]0.5 is therefore a property of the Delta9-80k large subunit. Autolysis at Ala9-Lys10 of the unmodified 80-kDa large subunit is not detectable on gels but was assayed by means of the fall in [Ca2+]0.5. This autolysis was complete in 3.5 min and was inhibited by high [NaCl]. The autolysis product of these calpains, which is essentially identical to that of natural m-calpain, was unstable in buffers of high ionic strength. Calpain in which the large subunit autolysis site had been mutated was fully active but did not undergo a drop in [Ca2+]0.5, showing that m-calpain is active prior to autolysis. The main physiological importance of autolysis of calpain is probably to generate an active but unstable enzyme, thus limiting the in vivo duration of calpain activity.