Permeabilization strategies to study protein phosphorylation.

This unit deals with the use of nucleotide triphosphates to label proteins in vitro in permeabilized cells and isolated cellular fractions. Both of these assay formats result in lysates from which the protein of interest may be easily immunoprecipitated; however alternative techniques are described for preparing the final lysate for electrophoretic analysis. A related procedure that does not involve permeabilization is outlined for direct analysis of cytosolic or membrane-bound kinases. Two different methods for determining the specific radioactivity of (32)P-containing compounds are also included. These experiments generally utilize [gamma-(32)P]ATP as an exogenously added phosphate donor, although [gamma-(32)P]GTP can be used in specific cases. The method is very straightforward, although numerous considerations must be made before applying it to each new system.

Assays of protein kinases using exogenous substrates.

In studies of the regulation of specific biochemical events by reversible phosphorylation, assaying the protein kinases themselves can often lead to significant progress in understanding the mechanistic details of a system under study. This unit describes assays for a variety of protein kinases that require different conditions to detect and measure their activities: cyclic nucleotide-dependent kinases, protein kinase C and isoforms, casein kinases, Ca(2+)/calmodulin-dependent kinases, and tyrosine kinase. A protocol for in-gel assays for specific kinase activity is also provided. The unit is not meant to be a catalog of individual protein kinase assays; however, the general principles of these assays should apply to most if not all known protein kinases.

Permeabilization strategies to study protein phosphorylation.

This unit deals with the use of nucleotide triphosphates to label proteins in vitro in permeabilized cells and isolated cellular fractions. These experiments generally utilize [g-(32)P]ATP as an exogenously added phosphate donor, although [g-(32)P]GTP can be used in specific cases. Procedures are outlined for performing a protein phosphorylation experiment using permeabilized cells and isolated intracellular organelles. Both of these procedures result in lysates from which the protein of interest may be easily immunoprecipitated; however alternative techniques are described for preparing the final lysate for electrophoretic analysis. A related procedure that does not involve permeabilization is outlined for direct analysis of cytosolic or membrane-bound kinases. Two different methods for determining the specific radioactivity of (32)P -containing compounds are also included.

Proximal biceps tendon rupture: primarily an injury of middle age.

A 51-year-old man suffered a rupture of the long-head tendon of the left biceps and a small rotator cuff tear while rock climbing. The typical signs and symptoms of a ruptured long-head biceps tendon include anterior shoulder pain, tenderness in the bicipital groove, and unusual bulging of the injured biceps. The history and physical exam are generally sufficient to make the diagnosis, but x-rays and MRI may be helpful to rule out related disorders. Because the injury is often associated with rotator cuff tendinitis, a complete shoulder exam is necessary. Conservative treatment will enable most patients to regain normal strength.

The uptake of Li+ into human 1321 N1 astrocytomas using 7Li NMR spectroscopy.

The uptake of Li+ ions into human 1321 N1 astrocytomas cultured on the surface of microcarrier beads was followed by 7Li NMR spectroscopy. The intracellular and extracellular 7Li resonances were separated by the use of dysprosium tripolyphosphate as a shift reagent. Excellent spectra were obtained from which the uptake of Li+ was found to be approximately ten times faster than that into human erythrocytes using the same technique and a steady-state intracellular Li+ concentration was observed within 60 min. The low intracellular Li+ concentration attained, relative to the extracellular concentration, indicates the presence of an efflux mechanism in astrocytomas that actively transports Li+ against its concentration gradient. The intracellular volume was estimated by quantitative 23Na NMR spectroscopy and the viability of the cells was confirmed by 31P NMR spectroscopy.

Inositol trisphosphate metabolism in Saccharomyces cerevisiae: identification, purification and properties of inositol 1,4,5-trisphosphate 6-kinase.

Ins(1,4,5)P3 metabolism was examined in Saccharomyces cerevisiae extracts. S. cerevisiae contains readily detectable Ins(1,4,5)P3 kinase activity that is predominantly soluble, but phosphomonoesterase activity acting on Ins(1,4,5)P3 was not detected in either soluble or particulate preparations from this organism. We have purified the kinase activity approximately 685-fold in a rapid four-step process, and obtained a stable preparation. The enzyme has an apparent native molecular mass of approximately 40 kDa, and displays Michaelis-Menten kinetics with respect to its two substrates, ATP and Ins(1,4,5)P3. The Km for ATP was 2.1 mM, and that for Ins(1,4,5)P3 was 7.1 microM. The enzyme appeared to be the first step in the conversion of Ins(1,4,5)P3 into an InsP5, and the partially purified preparation contained another activity that converted the InsP4 product into an InsP5. The InsP4 product of the partially purified kinase was not metabolized by human erythrocyte ghosts and co-chromatographed with an Ins(3,4,5,6)P4 [L-Ins(1,4,5,6)P4] standard, identifying it as D-Ins(1,4,5,6)P4. The yeast enzyme is thus an Ins(1,4,5)P3 6-kinase. This activity may be an important step in the production of inositol polyphosphates such as InsP5 and InsP6 in S. cerevisiae.

Phosphatidylinositol 3,4,5-trisphosphate is formed from phosphatidylinositol 4,5-bisphosphate in thrombin-stimulated platelets.

Platelets accumulate PtdIns(3,4,5)P3 and PtdIns(3,4)P2 in response to thrombin and thrombin-receptor-directed peptide in a GTP-dependent manner. These phosphoinositides are considered to be mediators of signaling events in a variety of cells. We have examined the metabolic route by which PtdIns(3,4,5)P3 and PtdIns(3,4)P2 are synthesized by briefly (10 min) incubating platelets with high activities of [32P]Pi, followed by 20 or 60 s exposure to thrombin, and analysing the relative radioactivities of the individual phosphate groups in the resulting labelled PtdIns(3,4,5)P3 and PtdIns(3,4)P2. The phosphate group possessing the highest specific activity under such non-equilibrium labelling conditions indicates the last one added in a metabolic sequence. The thrombin-stimulated rate of labelling of PtdIns(3,4)P2 was significantly slower than that of PtdIns(3,4,5)P3. Increased labelled PtdIns3P was not detected within 60 s. The measured relative radioactivities decreased in the order 3 > 5 > 4 >> 1 for PtdIns(3,4,5)P3 and 3 > 4 >> 1 for PtdIns(3,4)P2. On the basis of the results of both rate-of-labelling and specific radioactivity analyses we conclude that PtdIns(3,4,5)Pa is formed by 3-OH phosphorylation of PtdIns(4,5)P2, whereas PtdIns(3,4)P2, may be formed by 3-OH phosphorylation of PtdIns4P and/or dephosphorylation of PtdIns(3,4,5)P3. These findings point to the activation of phosphoinositide 3-kinase as a critical receptor-regulated step in thrombin-stimulated platelets.

The neuropeptide Phe-Met-Arg-Phe-NH2 (FMRFamide) increases levels of inositol 1,4,5-trisphosphate in the tentacle retractor muscle of Helix aspersa.

The C3 neurone, which acts as a motoneurone for the tentacle retractor muscle in Helix aspersa, contains both Phe-Met-Arg-Phe-NH2 (FMRFamide) and acetylcholine (ACh). Each of these transmitter substances evokes contraction of the isolated muscle. FMRFamide induces a delayed rise in tension followed by phasic contractions. Unlike the response to ACh, this response is not associated with a depolarization of the muscle cells. Here we show that FMRFamide stimulates the inositol phosphate second messenger system in the muscle and causes a significant increase in total inositol trisphosphate (InsP3) levels. The isomer which releases intracellular Ca2+ stores, inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), is increased in a similar proportion to the total InsP3. The production of Ins(1,4,5)P3 is therefore likely to be involved in the response of the muscle to FMRFamide and may account for the oscillatory nature of the mechanical response. The N-terminally extended heptapeptide pGlu-Asp-Pro-Phe-Leu-Arg-Phe-NH2 (pQDPFLRFamide), which relaxes the muscle, had no acute effect on InsP3 levels. Indirect evidence also indicates that intracellular Ca2+ stores are required for the generation of the FMRFamide response.

Phosphatidylinositol 3-kinase is activated by nerve growth factor and epidermal growth factor in PC12 cells.

The effects of nerve growth factor (NGF) and epidermal growth factor (EGF) on the regulation of phosphatidylinositol 3-kinase (PtdIns 3-kinase) activity were assessed in rat pheochromocytoma (PC12) cells. Both NGF and EGF induced a rapid activation of PtdIns 3-kinase as assessed by a dramatic rise in growth factor-induced PtdIns 3-kinase activity found in antiphosphotyrosine immunoprecipitates. The intracellular levels of two of the lipid products of PtdIns 3-kinase, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) and phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2), also rose dramatically, exhibiting time courses very similar to the appearance of PtdIns 3-kinase in immunoprecipitates. The activation of PtdIns 3-kinase is, therefore, a common event in the signal transduction processes elicited by growth factors stimulating distinct cellular end points in PC12 cells, namely the NGF-induced neuronal differentiation and EGF-stimulated mitogenesis. Thus the intracellular products of this enzyme may function in early biochemical events that are common components of the pathways controlling both differentiation and proliferation.

The stereoselective recognition of substrates by phosphoinositide kinases. Studies using synthetic stereoisomers of dipalmitoyl phosphatidylinositol.

Soluble phosphatidylinositol (PtdIns) 4- and 3-kinase activities were partially purified and characterized from human placental extracts. The placental PtdIns 4-kinase (type 3) has a Km for ATP of 460 microM and is kinetically different to a partially purified human erythrocyte, membrane-bound, PtdIns 4-kinase (type 2). These three inositol lipid kinases were then used to compare their substrate specificities against the four synthetic stereoisomers of dipalmitoyl PtdIns. Only the placental 4-kinase was influenced by the chirality of the glycerol moiety of PtdIns. However, neither of the 4-kinases was able to phosphorylate L-PtdIns and, therefore, these kinases have an absolute requirement for the inositol ring to be linked to the glyceryl backbone of the lipid through the D-1 position. Phosphoinositide 3-kinase, on the other hand, was found to phosphorylate both D- and L-PtdIns. While the 3-kinase phosphorylated exclusively the D-3 position of D-PtdIns, further analyses demonstrated that the same enzyme phosphorylated two sites on L-PtdIns, namely the D-6 and D-5 positions of the inositol ring. Some implications of these findings are discussed.

Phosphoinositide 3-kinase: a new effector in signal transduction?

Interest in phosphoinositide 3-kinase (PI 3-kinase) has been fuelled by its identification as a major phosphotyrosyl protein detected in cells following growth factor stimulation and oncogenic transformation. It is found complexed with activated growth factor receptors and non-receptor tyrosine kinases, thus suggesting that it participates in the signal transduction pathways initiated by the activation of tyrosine kinases. PI 3-kinase phosphorylates the 3-position in the inositol ring of the well known inositol phospholipids in vitro giving phosphatidylinositol 3-phosphate, phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate [PtdIns3P, PtdIns(3,4)P2 and PtdIns(3,4,5)P3], respectively. The cellular levels of PtdIns(3,4)P2 and PtdIns(3,4,5)P3 rapidly increase in circumstances where PI 3-kinase becomes complexed with tyrosine kinases. Accumulation of the same lipids also occurs in platelets and neutrophils following stimulation of G-protein linked alpha-thrombin and chemotactic peptide receptors, respectively, leading to speculation that one or both of these lipids is a new second messenger whose function is not yet known. This review brings together recent information on the isolation, characterization and regulation of PI 3-kinase, the cellular occurrence of 3-phosphorylated inositol phospholipids and possible functions of the PI 3-kinase pathway in cell signalling.

Age of the floor of the eastern Indian ocean.

Deep sea drilling in the eastern Indian Ocean shows that the oceanic crust off Western Australia is approximately 140 million years old and becomes younger to the west; this dates the initial opening of the Indian Ocean.