Synergistic activation of stress-activated protein kinase 1/c-Jun N-terminal kinase (SAPK1/JNK) isoforms by mitogen-activated protein kinase kinase 4 (MKK4) and MKK7.

Stress-activated protein kinase 1 (SAPK1), also called c-Jun N-terminal kinase (JNK), becomes activated in vivo in response to pro-inflammatory cytokines or cellular stresses. Its full activation requires the phosphorylation of a threonine and a tyrosine residue in a Thr-Pro-Tyr motif, which can be catalysed by the protein kinases mitogen-activated protein kinase kinase (MKK)4 and MKK7. Here we report that MKK4 shows a striking preference for the tyrosine residue (Tyr-185), and MKK7 a striking preference for the threonine residue (Thr-183) in three SAPK1/JNK1 isoforms tested (JNK1 alpha 1, JNK2 alpha 2 and JNK3 alpha 1). For this reason, MKK4 and MKK7 together produce a synergistic increase in the activity of each SAPK1/JNK isoform in vitro. The MKK7 beta variant, which is several hundred-fold more efficient in activating all three SAPK1/JNK isoforms than is MKK7 alpha', is equally specific for Thr-183. MKK7 also phosphorylates JNK2 alpha 2 at Thr-404 and Ser-407 in vitro, Ser-407 being phosphorylated much more rapidly than Thr-183 in vitro. Thr-404/Ser-407 are phosphorylated in unstimulated human KB cells and HEK-293 cells, and phosphorylation is increased in response to an osmotic stress (0.5 M sorbitol). However, in contrast with Thr-183 and Tyr-185, the phosphorylation of Thr-404 and Ser-407 is not increased in response to other agonists that activate MKK7 and SAPK1/JNK, suggesting that phosphorylation of these residues is catalysed by another protein kinase, such as CK2, which also phosphorylates Thr-404 and Ser-407 in vitro. MKK3, MKK4 and MKK6 all show a strong preference for phosphorylation of the tyrosine residue of the Thr-Gly-Tyr motifs in their known substrates SAPK2a/p38, SAPK3/p38 gamma and SAPK4/p38 delta. MKK7 also phosphorylates SAPK2a/p38 at a low rate (but not SAPK3/p38 gamma or SAPK4/p38 delta), and phosphorylation occurs exclusively at the tyrosine residue, demonstrating that MKK7 is intrinsically a 'dual-specific' protein kinase.

Serine 727 phosphorylation and activation of cytosolic phospholipase A2 by MNK1-related protein kinases.

We have previously reported that in thrombin-stimulated human platelets, cytosolic phospholipase A(2) (cPLA2) is phosphorylated on Ser-505 by p38 protein kinase and on Ser-727 by an unknown kinase. Pharmacological inhibition of p38 leads to inhibition of cPLA2 phosphorylation at both Ser-505 and Ser-727 suggesting that the kinase responsible for phosphorylation on Ser-727 is activated in a p38-dependent pathway. By using Chinese hamster ovary, HeLa, and HEK293 cells stably transfected with wild type and phosphorylation site mutant forms of cPLA2, we show that phosphorylation of cPLA2 at both Ser-505 and Ser-727 and elevation of Ca(2+) leads to its activation in agonist-stimulated cells. The p38-activated protein kinases MNK1, MSK1, and PRAK1 phosphorylate cPLA2 in vitro uniquely on Ser-727 as shown by mass spectrometry. Furthermore, MNK1 and PRAK1, but not MSK1, is present in platelets and undergo modest activation in response to thrombin. Expression of a dominant negative form of MNK1 in HEK293 cells leads to significant inhibition of cPLA2-mediated arachidonate release. The results suggest that MNK1 or a closely related kinase is responsible for in vivo phosphorylation of cPLA2 on Ser-727.

Evidence that 3-phosphoinositide-dependent protein kinase-1 mediates phosphorylation of p70 S6 kinase in vivo at Thr-412 as well as Thr-252.

Protein kinase B and p70 S6 kinase are members of the cyclic AMP-dependent/cyclic GMP-dependent/protein kinase C subfamily of protein kinases and are activated by a phosphatidylinositol 3-kinase-dependent pathway when cells are stimulated with insulin or growth factors. Both of these kinases are activated in cells by phosphorylation of a conserved residue in the kinase domain (Thr-308 of protein kinase B (PKB) and Thr-252 of p70 S6 kinase) and another conserved residue located C-terminal to the kinase domain (Ser-473 of PKB and Thr-412 of p70 S6 kinase). Thr-308 of PKBalpha and Thr-252 of p70 S6 kinase are phosphorylated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) in vitro. Recent work has shown that PDK1 interacts with a region of protein kinase C-related kinase-2, termed the PDK1 interacting fragment (PIF). Interaction with PIF converts PDK1 from a form that phosphorylates PKB at Thr-308 alone to a species capable of phosphorylating Ser-473 as well as Thr-308. This suggests that PDK1 may be the enzyme that phosphorylates both residues in vivo. Here we demonstrate that PDK1 is capable of phosphorylating p70 S6 kinase at Thr-412 in vitro. We study the effect of PIF on the ability of PDK1 to phosphorylate p70 S6 kinase. Surprisingly, we find that PDK1 bound to PIF is no longer able to interact with or phosphorylate p70 S6 kinase in vitro at either Thr-252 or Thr-412. The expression of PIF in cells prevents insulin-like growth factor 1 from inducing the activation of the p70 S6 kinase and its phosphorylation at Thr-412. Overexpression of PDK1 in cells induces the phosphorylation of p70 S6 kinase at Thr-412 in unstimulated cells, and a catalytically inactive mutant of PDK1 prevents the phosphorylation of p70 S6K at Thr-412 in insulin-like growth factor 1-stimulated cells. These observations indicate that PDK1 regulates the activation of p70 S6 kinase and provides evidence that PDK1 mediates the phosphorylation of p70 S6 kinase at Thr-412.

Identification of the separate domains in the hepatic glycogen-targeting subunit of protein phosphatase 1 that interact with phosphorylase a, glycogen and protein phosphatase 1.

Deletion and mutational analyses of the rat liver glycogen-targeting subunit (GL) of protein phosphatase 1 (PP1) have identified three separate domains that are responsible for binding of PP1, glycogen and phosphorylase a. The glycogen-binding domain spans the centre of GL between residues 144 and 231 and appears to be distinct from the glycogen-binding (storage) site of phosphorylase. The regulatory high-affinity binding site for phosphorylase a lies in the 16 amino acids at the C-terminus of GL. The PP1-binding domain is deduced to comprise the -RVXF- motif [Egloff, Johnson, Moorhead, Cohen and Barford (1997) EMBO J. 16, 1876-1887] located at residues 61-64 of GL and preceding lysine residues at positions 56, 57 and 59. A possible approach for increasing glycogen synthesis in certain disorders is discussed.

Cloning of a novel testis specific protein serine/threonine phosphatase, PPN 58A, from Drosophila melanogaster.

A gene encoding a novel member of the PPP family of protein serine/threonine phosphatases, termed PPN 58A, was cloned from Drosophila melanogaster. The deduced amino acid sequence of PPN 58A exhibits 59-62% identity to D. melanogaster PP1 isoforms, 51% identity to D. melanogaster PPY 55A and < or = 40% identity to other members of the PPP family. The single copy gene PPN 58A maps to chromosome 2 locus 58A. Analysis of PPN 58A mRNA reveals that, like PPY 55A, PPN 58A is a testis specific enzyme.

PPP1R6, a novel member of the family of glycogen-targetting subunits of protein phosphatase 1.

A complementary DNA encoding a novel human protein phosphatase 1 (PP1) glycogen-targetting subunit of molecular mass 33 kDa has been sequenced. PPP1R6 is 31% identical to the glycogen-targetting subunit (G(L)) of PP1 from rat liver, 28% identical to the N-terminal region of the glycogen-targetting subunit (G(M)) from human skeletal muscle and 27% identical to glycogen-targetting subunit PPP1R5. Unlike human PPP1R5 and its murine homologue PTG, whose mRNAs are most abundant in skeletal muscle, heart and liver, PPP1R6 is present at similar levels in a wide variety of tissues. The PPP1R6 is associated with glycogen in muscle but is not subject to the same modes of covalent and allosteric regulation as G(M) and G(L).

Deficiency of protein phosphatase 2A uncouples the nuclear and centrosome cycles and prevents attachment of microtubules to the kinetochore in Drosophila microtubule star (mts) embryos.

A Drosophila strain, carrying a P[lacW] element in the promoter of the protein phosphatase 2A (PP2A) catalytic subunit gene at chromosomal location 28D, has been identified using plasmid rescue of the P element and adjoining genomic DNA in Escherichia coli. Reversion mutagenesis was employed to demonstrate that the observed phenotype of the Drosophila strain was due to a single P[lacW] element insertion at 28D and to create three deficiency strains at this locus. Drosophila heterozygous for P[lacW]28D have reduced levels of PP2A mRNA and reduced PP2A catalytic activity against four different substrates compared to wild type, while homozygotes are deduced to have approximately 20% of wild-type PP2A activity. P[lacW]28D homozygotes, termed microtubule star (mts), die in embryo-genesis around the time of cellularisation, exhibiting over-condensed chromatin and a block in mitosis between prophase and the initiation of anaphase. Multiple centrosomes are visible in cellularised embryos, suggesting that PP2A may play a role in coupling the nuclear and centrosome cycles. When embryos arrest just prior to cellularisation, disorganised elongated arrays of microtubules radiate from centrosomes in all directions, but they are rarely associated with any DNA, suggesting that PP2A is required for the attachment of microtubules to chromosomal DNA at the kinetochore.

Reflex peripheral vasoconstriction is diminished in older men.

The purpose of this study was to compare reflex control of limb blood flow in healthy young (Y; 26 +/- 2 yr) and older (O;61 +/- 2 yr) men during whole body cooling under resting conditions. To better isolate the effect of chronological age, the two age groups (n = 6 per group) were closely matched for maximal oxygen uptake, body surface area, skinfold thickness, and fat-free weight. Subjects sat in an environmentally controlled chamber clad in standardized (0.6-clo) light cotton clothing at a dry-bulb temperature (Tdb) of 28 degrees C. After 30 min, Tdb was decreased by 2 degrees C every 5 min until Tdb = 10 degrees C, where it was held constant for the remainder of the 120-min session. Esophageal and mean skin temperatures were monitored continuously. Forearm blood flow (FBF) was measured every 5 min by venous occlusion plethysmography by using a mercury-in-Silastic strain gauge while arm temperature between the wrist and elbow was clamped at 37.2 +/- 0.1 degrees C by localized warm air heating. In this way, limb vasoconstriction was driven solely by thermoregulatory reflexes and not by direct effects of localized cooling. Mean skin temperature decreased at a similar rate and to a similar extent (by approximately 6 degrees C over a 2-h period) in both age groups, whereas esophageal temperature was relatively unaffected. In response to the local heating, the Y group maintained a significantly higher FBF than did the O group during the initial 30 min but decreased FBF during the cooling phase at a greater rate and to a greater extent than did the O group, leading to a significantly lower FBF during the final 30 min (at Tdb = 10 degrees C). Because there was no age difference in the mean arterial pressure response, similar effects of age were seen on forearm vascular conductance (FBF/mean arterial pressure). It was concluded that older men have a diminished reflex limb vasoconstrictor response to skin cooling. Furthermore, this difference in control of peripheral blood flow appears to be related to age per se; i.e., it is not a reflection of age-related differences in maximal oxygen uptake or body composition.

Drosophila PPY, a novel male specific protein serine/threonine phosphatase localised in somatic cells of the testis.

Drosophila protein phosphatase Y (PPY) displays 64% amino acid identity to protein serine/threonine phosphatase 1 (PP1) and 39% to protein phosphatase 2A (PP2A). Here we show by expression of cDNA in bacteria, that PPY is a protein serine phosphatase and that its biochemical properties are distinct from PP1 in both substrate specificity and regulation by the thermostable inhibitory proteins inhibitor 1 and inhibitor 2. We also demonstrate that PPY is a novel testis specific protein phosphatase by analysis of both mRNA and protein distribution. More precise immunolocalisation within the testis, using affinity purified anti-PPY protein and anti-PPY peptide antibodies, shows that PPY is present in somatic cyst cells, which encase the germ cells. The predominant location of PPY is in the nuclei of both head and tail cyst cells throughout the length of the testis except for the apical tip. The distribution of PPY, coupled with its unique biochemical properties, suggests that PPY may be required to prevent cyst cell division, increase transcription for provision of nutrients to the germ cells and/or provide a signal for spermatocyte differentiation.

Effects of age and acclimation on responses to passive heat exposure.

To examine the effect of chronological age on thermoregulation during passive heat exposure, six older (O, 61 +/- 1 yr) and six young (Y, 26 +/- 2 yr) men sat at rest during a 30-min baseline period (dry-bulb temperature = 28 degrees C), a 60-min thermal transient (28-46 degrees C by 2 degrees C steps every 5 min), and 30 min at 46 degrees C dry-bulb temperature. Subjects were matched for maximal O2 consumption, anthropometry, and body composition. Testing was repeated after a 9-day active heat acclimation protocol. There were no age differences in rectal (Tre), mean skin (Tsk), or mean body temperature (Tb = 0.8Tre + 0.2Tsk) before or after acclimation, but heart rate was lower (P < 0.01) in the O group in both acclimation states. Heat acclimation resulted in a significantly lower baseline Tre and Tb in both groups, which remained lower throughout the passive heat stress (P < 0.05). To examine the effects of age and acclimation on thermoregulatory effector function, forearm blood flow (by venous occlusion plethysmography) and chest sweating rate (SRch, by dew-point hygrometry) were plotted against Tb. The slope of the forearm blood flow-Tb relationship was significantly (P < 0.05) lower in the O group before and after acclimation. A lower maximal SRch (P < 0.05) was achieved by the O group, but neither the slope of SRch-Tb relationship nor the Tb threshold for sweating was affected by age. Predictably, acclimation resulted in a lower Tb threshold for the onset of sweating and skin vasodilation.(ABSTRACT TRUNCATED AT 250 WORDS)

Psychrometric limits to prolonged work in protective clothing ensembles.

When work is performed by workers in protective clothing, sweat evaporation is limited and body temperature rises. In an attempt to quantify the limits such ensembles place on safe work, 6 acclimated men and women walked at 30% VO2max (150-200 W/m2) in 2 protocols involving environmental transients. In one, ambient water vapor pressure (Pw) was fixed at 10 torr, and after rectal temperature (Tre) plateaued, ambient dry-bulb temperature (Tdb) was raised 2 degrees C every 10 min. In the second, Tdb was constant and Pw was increased 2 torr every 10 min. Critical temperature (Tcrit) and pressure (Pcrit) were defined as the Tdb or Pw at which thermal balance could no longer be maintained and Tre rose sharply. Each test was performed in various clothing ensembles ranging from light cotton work clothes to "impermeable" suits. Lines connecting mean Tcrit and mean Pcrit define a limit for safe prolonged exposure/exercise for approximately 50% of the population in each ensemble. Similar lines, drawn to represent values 2 standard deviations below the mean, should provide critical environmental limits for 95% of the population.

The effect of aerobic conditioning on venous pooling in the foot.

Nineteen fit college-age men were studied using foot (mid-arch) mercury-in-silastic strain gauge plethysmography before and after an 8-wk aerobic conditioning (running) program. Foot volume changes were followed through two maneuvers: a 15-s Trendelenburg procedure (passive leg elevation with subsequent relaxation in the dependent position) and a 15-s dynamic dorsi-flexion/plantar flexion exercise and subsequent relaxation. The conditioning regimen consisted of running 40 min, 3 d.wk-1 and resulted in a 10% (P less than 0.01) increase in VO2max. Following this regimen, subjects exhibited an increased blood volume drainage during the Trendelenburg procedure (mean delta VT = 3.3 ml.100 ml-1 pre-training, 3.8 ml.100 ml-1 post-training, P less than 0.05), but no significant change in delta VE (2.7 ml.100 ml-1 for all subjects). Muscle pump efficacy, defined as the ratio between delta VE and delta VT, did not change (64%). These data suggest that increased aerobic power via weight-bearing exercise training results in an increased foot venous pooling, but does not affect relative muscle pump function. This apparent increase in vascular pooling may be a physical response to the hypervolemia induced by endurance training, aiding in maintaining the constancy of vascular pressures.

Physiologically derived critical evaporative coefficients for protective clothing ensembles.

When work is performed in heavy clothing, evaporation of sweat from the skin to the environment is limited by layers of wet clothing and air. The magnitude of decrement in evaporative cooling is a function of the clothing's resistance to permeation of water vapor. A physiological approach has been used to derive effective evaporative coefficients (he) which define this ability to evaporate sweat. We refined this approach by correcting the critical effective evaporative coefficient (K for sweating efficiency (Ke,eta') since only a portion of the sweat produced under such conditions is evaporated through the clothing. Six acclimated men and women walked at 30% maximal O2 consumption (150-200 W.m-2) at a constant dry bulb temperature as ambient water vapor pressure was systematically increased 1 Torr every 10 min. Critical pressure was defined as the partial pressure of water vapor (Pw) at which thermal balance could no longer be maintained and rectal temperature rose sharply. Each test was performed in various clothing ensembles ranging from cotton shirt and pants to "impermeable" suits. This approach was used to derive he by solving the general heat balance equation, M - W +/- (R + C) = w.he.(Psk - Pw), where M is metabolic heat production, W is external work, R is radiant heat exchange, C is convective heat transfer, w is skin wettedness, and Psk is water vapor pressure of fully wet skin.(ABSTRACT TRUNCATED AT 250 WORDS)

An analysis of the unconfined compression of articular cartilage.

Analytical solutions have been obtained for the internal deformation and fluid-flow fields and the externally observable creep, stress relaxation, and constant strain-rate behaviors which occur during the unconfined compression of a cylindrical specimen of a fluid-filled, porous, elastic solid, such as articular cartilage, between smooth, impermeable plates. Instantaneously, the "biphasic" continuum deforms without change in volume and behaves like an incompressible elastic solid of the same shear modulus. Radial fluid flow then allows the internal fluid pressure to equilibrate with the external environment. The equilibrium response is controlled by the Young's modulus and Poisson's ratio of the solid matrix.

The mechanical properties of articular cartilage.

An outline of a theoretical model for the mechanical behavior of articular cartilage is presented. The model explicitly describes the pressures and flows generated in the interstitial fluid during loading and deformation. A number of experimental tests to measure the mechanical properties of cartilage are available. The interpretation of these test results in the light of this model and the important factors to consider in the design of experiments are reviewed.

Variations in the intrinsic mechanical properties of human articular cartilage with age, degeneration, and water content.

UNLABELLED: In a series of 103 specimens from the lateral facet of the human patella, the intrinsic mechanical properties of articular cartilage were measured using a confined compression creep test. By considering the cartilage as a porous, permeable solid filed with fluid, this experimental procedure allowed the determination of the intrinsic equilibrium modulus of the cartilage matrix and its permeability to fluid flow. The intrinsic equilibrium modulus and the permeability both were highly correlated with the water content of the tissue; as water content increased, the matrix of the tissue became softer and more permeable. There was only a marginal decrease in the equilibrium modulus of the tissue with increasing age and surface degeneration. The permeability of the cartilage matrix was not significantly correlated with age or degeneration. CLINICAL RELEVANCE: We concluded that the visual or histological appearance of a cartilage specimen may be a poor indicator of its ability to function as the bearing material in the intact joint. A more reliable indicator of the functional properties of a specimen can be obtained either by direct mechanical testing or by biochemical analysis of its composition.

Changes in the deformational behavior of human hip cartilage with age.

The deformation occurring in the articular cartilage covering the human femoral head has been measured both when the femoral head is loaded in its natural acetabulum and when the cartilage is loaded with a small indentor. The results indicate that the material response is substantially different in these two situations. In the intact joint the cartilage deformation is substantially greater in older joints, but the response of cartilage to loading with an indentor does not change significantly with age. Theoretical elastic models of the cartilage behavior in these two situations were analyzed. For old cartilage which is idealized as an elastic material the increased deformation which is observed in the intact joint can be attributed to changes in Poisson's ratio, though in the real material increased fluid flux under load is the more probable cause.