Gary E. Landreth

Phosphorylation of Insulin Receptors Solubilized from Rat Skeletal Muscle

A method has been developed to solubilize insulin receptors from skeletal muscles. Rat hindlimb muscles were rapidly frozen in liquid nitrogen, powdered, extracted with buffered Triton X-100, and partially purified by differential centrifugation followed by wheat germ agglutinin affinity chromatography. The solubilized receptors exhibit typical curvilinear Scatchard plots in insulin binding assays: rapid, Mn2+-dependent autophosphorylation of the β-subunit on exposure to insulin as well as insulin-stimulated kinase activity toward histone H2B. Furthermore, when intact soleus muscles were incubated in phosphate-depleted medium containing Na2H[32P]PO4, addition of insulin stimulated the in situ phosphorylation of the β-subunit of the insulin receptor. The ability to rapidly and efficiently isolate insulin receptors from skeletal muscle may permit investigation of factors that modulate insulin action in this tissue.

Selective Inhibition of Nerve Growth Factor-Stimulated Protein Kinases by K-252a and 5′-S-Methyladenosine in PC12 Cells

K‐252a, a protein kinase inhibitor isolated from the culture broth of Nocardiopsis sp., inhibits the nerve growth factor (NGF)‐stimulated phosphorylation of microtubule‐associated protein 2 (MAP2) and Kemptide (synthetic Leu‐Arg‐Arg‐Ala‐Ser‐Leu‐Gly) by blocking the activation of two independent kinases in PC 12 cells: MAP2/pp250 kinase and Kemptide kinase. The NGF‐stimulated activation of these kinases is inhibited in a dose‐dependent manner following treatment of the cells with K‐252a. Although these kinases also are activated by epidermal growth factoi (EGF) and 12‐O‐tetradecanoyl‐phorbol 13‐acetate, K‐252a has no inhibitory effect when these agents are used. Half‐maximal inhibition of the activation of both kinases was observed at 10–30 nM K‐252a. K‐252a was shown to directly inhibit the activity of MAP2/pp250 kinase and Kemptide kinase when added tc the phosphorylation reaction mixture in vitro; however, half‐maximal inhibition under these conditions was observed at ±50 nM K‐252a. These data suggest that K‐252a exerts its effects at a step early in the cascade of events following NGF binding. The effects of K‐252a are similar to those reported for 5′‐S‐methyladenosine (MTA) and other methyltransferase inhibitors. Treatment of PC12 cells with MTA inhibited NGF‐, but not EGF‐mediated activation of MAP2/pp250‐kinase (Ki± 500 μM). MTA, when added to the phosphorylation reaction mixture in vitro, directly inhibited kinase activity (Ki= 50 μM), suggesting that the effects of MTA may be the result of its action on protein kinases rather than methyltransferases.

Characterization of a Nerve Growth Factor-Stimulated Protein Kinase in PC12 Cells Which Phosphorylates Microtubule-Associated Protein 2 and pp250

Treatment of PC12 cells with nerve growth factor (NGF) resulted in the rapid, but transient, activation of a protein kinase which specifically phosphorylated an endogenous 250‐kDa cytoskeletal protein (pp250). We report that the microtubule‐associated protein, MAP2, is an alternative substrate for the NGF‐activated kinase. NGF treatment maximally activated the kinase within 5 min; however, the activity declined with longer exposure to NGF. The enzyme was localized predominantly in microsomal and soluble fractions and phosphorylated MAP2 on serine and threonine residues. The soluble enzyme was fractionated by DEAE chromatography and gel filtration and had an apparent Mr of 45,000. The enzyme was purified to near homogeneity by chromatofocussing and had a pl of 4.9. Kinetic analysis revealed that NGF treatment caused a sevenfold increase in Vmax for MAP2. The Km with respect to the MAP2 substrate was approximately 50 nM and was not altered by NGF treatment. A novel feature of the NGF‐stimulated enzyme was its sharp dependence on Mn2+ concentration. The active enzyme is likely to be phosphorylated, because inclusion of phosphatase inhibitors was required for recovery of optimal activity and the activity was lost on treatment of the enzyme with alkaline phosphatase. Histones, tubulin, casein, bovine serum albumin, and the ribosomal subunit protein S‐6 were not phosphorylated by this enzyme. The NGF‐stimulated kinase was distinct from A kinase, C kinase, or other NGF‐stimulated kinases. The rapid and transient activation of the protein kinase upon NGF treatment suggests that the enzyme may play a role in signal transduction in PC12 cells.

Enhanced tyrosine phosphorylation in B lymphocytes upon complexing of membrane immunoglobulin

Activation of normal human peripheral blood B-enriched lymphocytes with antiserum reacting with membrane immunoglobulin was associated with increased phosphorylation of cellular proteins, particularly in the Triton-insoluble cytoskeletal fraction. At least two proteins (56K and 60K) were labeled predominantly at tyrosine residues. These findings may indicate another specialized membrane structure which upon specific ligand binding stimulates tyrosine phosphorylation in association with cellular proliferation.

Nerve growth factor stimulates the phosphorylation of a 250 kDa cytoskeletal protein in cell-free extracts of PC12 cells

Nerve growth factor (NGF) rapidly stimulates the phosphorylation of a 250 kDa cytoskeletally-associated protein (pp250) by a protein kinase which is also associated with structural elements of the cell. We have solubilized these proteins and demonstrated that NGF-stimulated phosphorylation can be observed in cell free extracts of cytoskeletons from NGF-treated PC12 cells. The pp250 substrate and the 250-kinase were solubilized from PC12 cytoskeletons by treatment with 2 M urea. Phosphorylation of pp250 was maximally stimulated following treatment of the cells for 5 min with NGF. This effect was transient, diminishing with longer exposure of the cells to hormone. The 250-kinase preferred Mn2+ over Mg2+ and was inhibited by both Na+ and K+. The phosphorylation of pp250 was not affected by Ca2+. Upon fractionation of the urea-soluble cytoskeletal proteins by gel filtration, the 250-kinase eluted in two peaks; one peak of enzyme activity coeluting with the pp250 substrate, and a second peak of enzyme activity eluting with an apparent Mr of approximately 60 kDa. Treatment of the PC12 cells with the phorbol ester TPA also stimulated the phosphorylation of pp250, although this effect was not as great as that produced by NGF. This cell free system should be a valuable tool in the investigation of the mechanisms of NGF action.

Expression of pp60c-src in adult and developing rat central nervous system

Brains of adult and fetal (E13-E19) rats were assayed by region for the presence of the proto-oncogene product pp60c-src. pp60c-src was abundant in the adult brain with the highest levels found in the cerebral hemispheres and localized to the cortical cellular layers. In the embryonic nervous system the levels of pp60c-src activity were much higher throughout the brain than those observed in the adult. The expression of pp60c-src was developmentally regulated, but demonstrated a regionally distinct pattern of expression. In the cortex src activity steadily rose during gestation, while in the basal forebrain and midbrain maximal activity was observed at E17 which then declined to adult levels. The data demonstrate that pp60c-src is differentially expressed in regions of the brain, both during development and in the adult.

Coordinate release of myosin and a high molecular weight microtubule-associated protein from PC12 cytoskeletons by ATP

The association of two high molecular weight (HMW) structural proteins with the cytoskeletons of rat pheochromocytoma cells, PC12, is regulated by ATP and other nucleotides. Exposure of PC12 cytoskeletons to ATP resulted in the selective solubilization of two HMW proteins, identified as myosin and a 280 kD microtubule-associated protein. These two proteins were rapidly released from the cytoskeleton following incubation with ATP, GTP, CTP, and ADP; non-hydrolysable ATP analog caused protein release to a less marked extent. The effect of the latter two nucleotides indicated that the release of the myosin and the HMW microtubule-associated protein was likely to be the result of nucleotide-induced conformational changes in one or both proteins. Myosin and the HMW microtubule-associated proteins interact with actin in vitro in a nucleotide-sensitive manner. The present data demonstrate that similar interactions are likely to exist within the intact cytoskeleton and suggest that the associations of these structural proteins with the cytoskeleton are regulated by common mechanisms. The results also suggest that the cells may differentially regulate the stability of a subset of these structural proteins in their interactions with other cytoskeletal elements.