BiP is a substrate for src kinase in vitro.

In a study to investigate the ability of chaperones to modulate src kinase activity, it was observed that BiP, a member of the HSP70 family found in the endoplasmic reticulum, is an excellent substrate for src kinase in vitro. The reaction requires polylysine and the results suggest that two tyrosine residues are phosphorylated. Although there is no evidence for this reaction in vivo, it does provide a very efficient method to label BiP.

Phosphorylation of tau protein by purified p34cdc28 and a related protein kinase from neurofilaments.

It has been suggested that hyperphosphorylation of the tau protein in neurofibrillary tangles may be relevant to the etiology of Alzheimer's disease and that at least one of the hyperphosphorylated sites lies within a consensus sequence for the p34cdc2/cdc28 family of kinases. We describe a new method for large-scale purification of p34cdc28 kinase from Saccharomyces cerevisiae and show that the purified enzyme can phosphorylate bovine and human tau. Phosphorylation was greatly enhanced by the addition of basic and acidic substrate modulators. The effect of the substrate modulators differed both with the structures of the substrates and the modulators. Similar results were obtained with a kinase that could be purified from neurofilaments by p13suc1 affinity chromatography, a hallmark of p34cdc2/cdc28-type kinases. These results are consistent with the hypothesis that a kinase of this type is involved in tau phosphorylation in vivo and open the possibility that hyperphosphorylation in Alzheimer's disease may be controlled by substrate modulators.

Heparin stimulates epidermal growth factor receptor-mediated phosphorylation of tyrosine and threonine residues.

We have described previously that in extracts of A431 cells epidermal growth factor (EGF) stimulates the phosphorylation of tyrosine as well as of threonine residues in the EGF receptor and in lipocortin 1. We now report that heparin at low concentrations also stimulates the autophosphorylation of the EGF receptor and of the recombinant 56-kDa domain of the EGF receptor that lacks the EGF binding site. To study the stimulations of phosphorylation of threonine residues, a fusion protein was prepared with glutathione S-transferase (GST) and an EGF receptor fragment, TK8 (residues 647-688), that contains the threonine phosphorylation site but no tyrosine. We show that the phosphorylation of threonine residues in GST-TK8 by extracts of A431 cells is stimulated by heparin but not by EGF. These and other results suggest that heparin acts as a chaperone, a substrate modulator, that enhances the susceptibility of the substrate to phosphorylation by protein kinases.

Regulation of 6-phosphofructo-1-kinase activity in ras-transformed rat-1 fibroblasts.

Fructose 2,6-bisphosphate (F-2,6-P2) stimulated glycolysis in cell-free extracts of both normal and ras-transfected rat-1 fibroblasts. The extract of the transformed cell glycolyzed more rapidly in both the absence and the presence of F-2,6-P2 than the extract of the parent fibroblast. Addition of mitochondrial ATPase (F1) or inorganic phosphate (Pi) further stimulated lactate production in both cell lines. F-2,6-P2 stimulated the 6-phosphofructo-1-kinase (PFK-1) activity in extracts of normal and transfected cells. The activity in extracts of transformed cells tested with a fructose 6-phosphate regenerating system was considerably higher than in the extract of normal cells. Stimulation of PFK-1 activity by cAMP of both cell lines was not as pronounced as that by F-2,6-P2. In the absence of F-2,6-P2 the PFK-1 activity was strongly inhibited in the transformed cell by ATP concentrations higher than 1 mM, whereas in the normal cell only a marginal inhibition was noted even at 2 or 3 mM ATP. F-2,6-P2 reversed the inhibition of PFK-1 by ATP. Nicotinamide adenine dinucleotide (NAD) at 100 microM (in the presence of 2 mM ATP and 1 microM F-2,6-P2) stimulated PFK-1 activity only in the transformed cell, whereas nicotinamide adenine dinucleotide phosphate (NADP) inhibited PFK-1 activity (in the presence or absence of 1 microM F-2,6-P2) in extracts of both cell lines. No previous observations of stimulation or inhibition by NAD or NADP on PFK-1 activity appear to have been reported. A threefold increase in the intracellular concentration of F-2,6-P2 was observed after transfection of rat-1 fibroblast by the ras oncogene. We conclude from these data that the PFK-1 activity of ras-transfected rat-1 fibroblasts shows a greater response to certain stimulating and inhibitory regulating factors than that of the parent cell.

Multiple changes induced by cholera toxin contribute to the stimulation of aerobic lactate production in rat-1 fibroblasts.

Exposure of rat-1 fibroblasts to cholera toxin increased aerobic lactate production 3- to 8-fold with maximal stimulation observed between 1 and 2 h at a concentration of 1-2 micrograms/ml. Concomitant with this change was a 10- to 40-fold elevation in the intracellular concentration of cAMP. The cell permeable cAMP analogue, N6,2'-O-dibutyryl cAMP and the cyclic nucleotide phosphodiesterase inhibitor RO-20-1724 also increased lactate production and intracellular cAMP levels, although less effectively. Cholera toxin and dibutyryl cAMP induced a 2- to 3-fold elevation of intracellular fructose 2,6-bisphosphate and 2- to 3-fold increases in both 3-O-methylglucose and inorganic phosphate transport. A survey of five additional cell lines revealed striking variabilities in their individual responses to cholera toxin and dibutyryl cAMP. All were observed to be considerably less sensitive to either agent than rat-1 cells. These data suggest that a cooperative effect involving multiple parameters may be responsible for the observed increases in aerobic lactate production in response to cAMP and that these parameters may vary significantly among cell lines.

Control of src kinase activity by activators, inhibitors, and substrate chaperones.

The activities of src tyrosine kinases are greatly influenced by substrate modulators (chaperones). In the presence of bovine serum albumin, the phosphorylation of a random polymer of glutamic acid, alanine, and tyrosine (1:1:1) by src kinases is stimulated 20- to 100-fold, but there is little stimulation with a polymer of glutamic acid and tyrosine (4:1) as substrate. This suggests that serum albumin interacts with the substrates rather than with the enzyme. groEL and several other heat shock proteins also stimulate the phosphorylation of a random polymer of glutamic acid, alanine, and tyrosine (1:1:1). In the absence of substrate modulators, the phosphorylation of calmodulin and of several ras proteins by src kinase is barely detectable. In the presence of polylysine or protamine, marked phosphorylation is observed. Another type of control of src kinase activities appears to be directed toward the enzyme rather than the substrate. Triton X-100 extracts of plasma membranes of bovine brain contain a heat-stable factor that stimulates c-src kinase activity with any of the polymers as substrate. The same extract contains a heat-labile factor that preferentially inhibits c-src kinase activity. The two factors are separated by DEAE-Sephacel and phosphocellulose chromatography. The presence of the activator enhances the potency of the inhibitor.

Stimulation of phosphorylation of lipocortin at threonine residues by epidermal growth factor (EGF) and the EGF receptor: addition of protein kinase P with polylysine inhibits this effect.

In this paper we show that epidermal growth factor (EGF) stimulates the phosphorylation of lipocortin 1, at threonine as well as at tyrosine residues, by a highly purified preparation of the EGF receptor. The phosphorylation of threonine residues is catalyzed by an enzyme that contaminates the receptor preparations, since crude extracts of A431 plasma membranes contain larger amounts of the threonine kinase than does the receptor preparation. Protein kinase P (2.5 ng) inhibits both threonine and tyrosine phosphorylation of lipocortin 1 while greatly stimulating the autophosphorylation of the EGF receptor. Acetyllipocortin 1 is poorly phosphorylated at tyrosine residues by the EGF receptor kinase, but it becomes readily phosphorylated in the presence of polylysine. The most likely explanation for this observation is that there is an interaction between polylysine and acetyllipocortin that converts the latter into a suitable substrate for the EGF receptor. These and other experiments described in this paper point to a role of surface charges in the susceptibility of substrates to attach by protein kinases.

Effect of chemically well-defined sphingosine and its N-methyl derivatives on protein kinase C and src kinase activities.

In view of the possible effects of the sphingoid base on protein kinases, and the fact that the sphingoid bases used in previous studies were not chemically well-defined, we have studied the effects of chemically well-defined sphingosines and their derivatives on kinase activity. Both (4E)-D- and (4E)-L-erythro-sphingenine showed a weak inhibitory effect, and (4E)-L-threo-sphingenine had a moderate inhibitory effect. In contrast, (4E)-N,N-dimethyl-D-erythro-sphingenine and the sphingosine preparation from a commercial source showed a strong inhibitory effect on PK-C in A431 cells as well as on purified PK-C. Synthetic (4E)-D-erythro-sphingenine and several samples of natural sphingosine inhibited v-src or c-src tyrosine kinase activity measured with polyglutamate-tyrosine (4:1) as substrate. N-Acetylated or N-methylated sphingosines did not inhibit src kinase activity, but rather produced a consistent 1.5-2-fold stimulation of such activity.

Decreased susceptibility of a 70-kDa protein to cathepsin L after phosphorylation by protein kinase C.

A 70-kDa protein is phosphorylated in cell-free preparations from rat or mouse fibroblasts by an endogenous protein kinase. This protein is immunologically related to a group of 68-kDa to 87-kDa proteins described in the literature as substrates for protein kinase C (PK-C). Although the phosphorylation of the 70-kDa protein by isolated plasma membranes takes place in the presence of EGTA, we conclude that the reaction is catalyzed by PK-C based on its inhibition by staurosporin. As shown previously, pure PK-C phosphorylates a synthetic random polymer of arginine and serine in the absence of Ca2+ and lipids, a reaction markedly stimulated by an endogenous unidentified activator of PK-C. When the 70-kDa protein from normal fibroblasts was exposed to the cytosol of chemically or ras-transformed fibroblasts, it disappeared as measured by phosphorylation by added PK-C. Cytosol of normal fibroblasts was much less effective (ca. 20%). Cathepsin L purified from rat kidney or from the medium of transformed cells had an effect similar to that of the cytosol of transformed cells. When the 70-kDa protein was phosphorylated by PK-C prior to exposure to cathepsin L or to the cytosol of transformed cells, there was a marked protection of the 70-kDa protein. We conclude that the 70-kDa protein is degraded by cathepsin L as ascertained by both immunological and biochemical assays and that it is protected by prior phosphorylation with PK-C. The possible role of this effect in signal transduction is discussed.

A view of misconduct in science.

KIE: An eminent biochemist gives his personal view of misconduct in science, one largely based on an experience with the case of fraud by a young researcher, Mark Spector, in Racker's own laboratory at Cornell University. He considers four general aspects of the problem of research fraud: the scientists involved, their supervisors and collaborators, the universities and funding agencies, and the federal government as represented by Congress. Racker argues that fraud committed by talented professional scientists springs from an unbalanced mind, that each case must be handled individually and pursued in the courts if the evidence warrants it, and that Congress must be persuaded that the research community can and will assume responsibility for the detection and punishment of scientific misconduct.^ieng

Brain protein kinase C phosphorylating poly(arginine,serine) or lamin B is stimulated by anions and by an activator purified from bovine serum albumin preparations.

The phosphorylation of histone by purified protein kinase C (PK-C) from rat brain is dependent on the presence of Ca2+ and lipids. Phosphorylation of a synthetic random polymer of arginine and serine (3:1) is only moderately enhanced by Ca2+ and lipids, but it is greatly enhanced in the absence of Ca2+ and lipids by a contaminant in crystalline bovine serum albumin or by heated cellular fractions. The phosphorylation ratio of histone to poly(arginine,serine) varies between different PK-C fractions from brains of rat, pig, or lamb. These variations are partly caused by a PK-C isozyme that prefers poly(arginine,serine) over histone as substrate. The kinase activator (KA) was partly purified from bovine serum albumin and from extracts of plasma membranes of human placenta. KA is also present in mitochondria, nuclei, and the cytosol. Sulfates and phosphates at 10 mM substitute for KA with poly(arginine,serine) as substrate. The phosphorylation of histone III in the presence of Ca2+ and lipids is moderately stimulated by KA, but the phosphorylation of lamin B and some other endogenous proteins is greatly enhanced by KA. With histones as substrates, inorganic anions do not stimulate phosphorylation. The phosphorylation of poly-(arginine,serine) is very sensitive to low concentrations of staurosporin and is inhibited by PK-C antibody, but, in contrast to histone phosphorylation, it is resistant to sphingosine and polymyxin B. The poly(arginine,serine) phosphorylating activity is more stable at 4 degrees C than the histone phosphorylating activity, but the latter is stabilized by 0.05% Triton X-100.

Altered sensitivity of system A amino acid transport to ouabain in normal and transformed C3H-10T1/2 cells during the cell cycle.

Quiescent C3H-10T1/2 mouse fibroblasts that have not undergone any type of stress have a relatively low rate of 2-aminoisobutyrate (Aib) uptake by means of system A, which is primarily energized by the transmembrane Na+ chemical gradient potential. System A activity in these cells is not sensitive to ouabain or proton ionophores. In contrast, methylcholanthrene-transformed and confluent C3H-10T1/2 cells treated with 0.4 mM ouabain for 16-20 hr utilize the membrane potential generated by the Na+, K+-ATPase pump to drive Aib transport by means of system A as shown by the sensitivity of transport activity to ouabain and proton ionophores. Since glucose is present during the assay, the proton ionophores do not affect the availability of ATP, as indicated by the undiminished uptake of 86Rb+ by the Na+, K+-ATPase pump. As cells progress through the G1 phase of the cell cycle, they show an increased system A activity prior to entry into the S phase, which is also dependent on the electrogenicity of the Na+, K+-ATPase pump. There appears to be in all these cases a qualitative shift in the bioenergetic mechanism for the uptake of Aib as well as a marked quantitative increase in Aib uptake. The high activity after ouabain treatment was sustained in the transformed cells after removal of the ouabain, whereas in the confluent 10T1/2 cells the rate of uptake decayed rapidly, suggesting a difference in the mode of regulation. We conclude that transformed cells and normal cells in late G1 or under stress make use of the membrane potential generated by the Na+, K+-ATPase pump to drive amino acid uptake by means of system A.

Purification of a vanadate-sensitive ATPase from clathrin-coated vesicles of bovine brain.

Clathrin-coated vesicle acidification is mediated by an N-ethylmaleimide-sensitive, vanadate-resistant proton-translocating ATPase. This enzyme is a 530-kDa hetero-oligomer which catalyzes ATP-dependent proton pumping when reconstituted (Xie, X. S., and Stone, D. K. (1986) J. Biol. Chem. 261, 2492-2495). We now report the purification of a second ATPase from bovine brain clathrin-coated vesicles which is inhibited by both N-ethylmaleimide (1 mM) and vanadate (10 microM). Localization of the ATPase to clathrin-coated vesicles was demonstrated by the precipitation of ouabain-resistant, vanadate-sensitive ATPase activity with anti-clathrin antibodies. The enzyme was solubilized with 0.1% polyoxyethylene 9-lauryl ether and has been purified 700-fold to a specific activity of 42 mumol of Pi.mg of protein-1.min-1. A molecular mass of 116 kDa was determined by centrifugation in sucrose gradients prepared in H2O and D2O, by high performance liquid chromatography using gel filtration, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis performed under reducing conditions. The ATPase is unlike any known mammalian E1E2-type ATPase in that it is not inhibited by ouabain or [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) and it is not activated by Na+, K+, or Ca2+.

Stimulation of inositol incorporation into lipids of PC12 cells by nerve growth factor and bradykinin.

The effects of bradykinin (BK) and lithium on the phosphatidylinositol cycle were examined in PC12 cells cultured for 20 h in the presence [PC12(+)] or in the absence [PC12(-)] of nerve growth factor (NGF). BK (1 microM) induced a small stimulation of the incorporation of myo-[2-3H]inositol into the lipids of PC12(-) cells and a three- to fourfold stimulation of such incorporation into the lipids of PC12 (+) cells. About 15 h of incubation with NGF and greater than 10 min of incubation with BK were needed for maximal stimulation of inositol incorporation by BK. In the presence of 25 mM LiCl, BK stimulated the inositol monophosphate levels nine-fold in PC12 (-) and 30-fold in PC12 (+) cells. After incubation for 20 h with NGF, an increased binding of [3H]BK to the PC12 (+) cells was observed at 4 degrees C. Exposure of the cells for 30 min to 25 mM LiCl enhanced the effect of BK on the inositol incorporation into total inositol lipids, especially in PC12(+) cells. In these cells, LiCl in the presence of BK also increased several-fold the intracellular levels of inositol bisphosphate and inositol trisphosphate.

Lithium stimulation of membrane-bound phospholipase C from PC12 cells exposed to nerve growth factor.

LiCl stimulated the formation of inositol monophosphate in PC12 cells that had been exposed to nerve growth factor (NGF) for 4-5 days. Half-maximal accumulation was observed at approximately 8 mM LiCl. Stimulation of formation of inositol bisphosphate plus inositol trisphosphate was half-maximal at approximately 1 mM LiCl. With membranes isolated from PC12 cells differentiated with NGF, the hydrolysis of added phosphatidylinositol 4,5-bisphosphate (PIP2) was stimulated by LiCl in a biphasic manner, with the first stimulation half-maximal at approximately 0.7 mM and the second half-maximal at approximately 15 mM LiCl. The apparent Km for PIP2 was lowered in the presence of 1.1 mM LiCl from approximately 200 to approximately 70 microM. Membranes from cells grown in the absence of NGF did not respond to LiCl. Although observations with intact cells are difficult to interpret without ambiguity, the results obtained with isolated membranes support our interpretation of the stimulatory action of lithium in the intact PC12 cells.

Motility, heat, and lactate production in ejaculated bovine sperm.

Effects of various inhibitors on motility, heat, and lactate production of ejaculated bovine sperm were determined in the presence of antimycin A and rotenone. erythro-9-[3-(2-Hydroxynonyl)]adenine (EHNA) and polyvinylpyrrolidone (PVP-360) stopped motility and reduced heat or lactate production by 30-50%. Carbodiimides resulted in loss of motility and a reduction of metabolism by 60-75%. Quercetin treatment, which enhanced rather than inhibited motility, depressed heat and lactate production by 50-60%. Since mechanical immobilization reduced heat production by only 30%, the question arises as to what other cellular processes are major contributors to the energy budget. Inhibitors of ion flux had little-to-no effect on heat or lactate production, suggesting that neither mitochondrial nor Na+/K+ ATPases were major ATP-requiring processes. Calcium flux at the plasma membrane also was minimal and previous reports eliminated glycolytic substrate cycling as major consuming processes for ATP. Although quercetin inhibited lactate production in intact cells, no effect of quercetin on cell-free glycolysis and the ATPase activities of isolated dynein was detected. Quercetin did, however, inhibit ATPase activity of plasma membrane, suggesting that this unidentified ATPase may contribute to the formation of ADP and Pi required for lactate production by the intact cell. We propose (a) that the bioenergetic costs of motility are divided between regulatory events and dynein-microtubule interaction (dynein ATPase), (b) that some of the membrane-related processes may be "inefficient," and (c) that quercetin may render these steps more "efficient," in a manner analogous to its action on the Na+/K+ pump of Ehrlich ascites tumor cells.