Tyrosine-specific phosphorylation of calmodulin by the insulin receptor kinase purified from human placenta.

It has previously been demonstrated that calmodulin can be phosphorylated in vitro and in vivo by both tyrosine-specific and serine/threonine protein kinase. We demonstrate here that the insulin receptor tyrosine kinase purified from human placenta phosphorylates calmodulin. The highly purified receptors (prepared by insulin-Sepharose chromatography) were 5-10 times more effective in catalysing the phosphorylation of calmodulin than an equal number of partially purified receptors (prepared by wheat-germ agglutinin-Sepharose chromatography). Phosphorylation occurred exclusively on tyrosine residues, up to a maximum of 1 mol [0.90 +/- 0.14 (n = 5)] of phosphate incorporated/mol of calmodulin. Phosphorylation of calmodulin was dependent on the presence of certain basic proteins and divalent cations. Some of these basic proteins, i.e. polylysine, polyarginine, polyornithine, protamine sulphate and histones H1 and H2B, were also able to stimulate the phosphorylation of calmodulin via an insulin-independent activation of the receptor tyrosine kinase. Addition of insulin further increased incorporation of 32P into calmodulin. The magnitude of the effect of insulin was dependent on the concentration and type of basic protein used, ranging from 0.5- to 9.0-fold stimulation. Maximal phosphorylation of calmodulin was obtained at an insulin concentration of 10(-10) M, with half-maximal effect at 10(-11) M. Either Mg2+ or Mn2+ was necessary to obtain phosphorylation, but Mg2+ was far more effective than Mn2+. In contrast, maximal phosphorylation of calmodulin was observed in the absence of Ca2+. Inhibition of phosphorylation was observed as free Ca2+ concentration exceeded 0.1 microM, with almost complete inhibition at 30 microM free Ca2+. The Km for calmodulin was approx. 0.1 microM. To gain further insight into the effects of basic proteins in this system, we examined the binding of calmodulin to the insulin receptor and the polylysine. Calmodulin binds to the insulin receptor in a Ca2+-dependent manner, whereas it binds to polylysine seemingly by electrostatic interactions. These studies identify calmodulin as a substrate for the highly purified insulin receptor tyrosine kinase of human placenta. They also demonstrate that the basic proteins, which are required for insulin to stimulate the phosphorylation of calmodulin, do so by a direct interaction with calmodulin.

Chelation of intracellular calcium prevents stimulation of glucose transport by insulin and insulinomimetic agents in the adipocyte. Evidence for a common mechanism.

Vanadate, concanavalin A (Con A), H2O2, and the phorbol ester, 12-O-tetradecanoyl phorbol 13-acetate (TPA), seemingly unrelated compounds, have effects on cellular metabolism similar to those of insulin. The mechanism(s) by which these diverse compounds act and their relevance to insulin action remain unclear. The hypothesis that increased intracellular calcium ([Ca2+]i) may provide the common basis for the stimulatory effects of these agents on glucose uptake in adipocytes was tested. This was accomplished by preincubating cells with the membrane-permeant ester, quin2-AM (2-[(2-bis[carboxymethyl] amino-5-methylphenoxy]-6-methoxy-8-bis[carboxymethyl]- aminoquinolinetetrakis [acetoxymethyl] ester), which is rapidly accumulated and hydrolyzed by esterases to form the impermeant tetracarboxylate chelator form, quin2. Vanadate, Con A, H2O2, and TPA stimulate D-glucose uptake in adipocytes approximately 3-, 3-, 2-, and 0.6-fold, respectively, compared to the 5- to 10-fold stimulation of D-glucose uptake obtained with insulin. Preincubation with quin2-AM produced a dose-dependent inhibitory effect only on the stimulated portion of glucose transport without affecting basal (unstimulated) transport. The concentrations for half-maximal inhibition (IC50) of stimulated glucose transport by quin2-AM were 26, 35, 25, 14, and 34 microM for insulin, vanadate, Con A, H2O2, and TPA, respectively. Quin2-AM maximally inhibited stimulated glucose uptake by greater than 85% for all of the insulin mimetic agents. In contrast the maximal inhibition of insulin-stimulated glucose transport by quin2-AM was 55 +/- 4%. Therefore, stimulation of glucose transport by insulin and other diverse compounds appears to involve at least one common calcium-dependent intermediate step.

The insulin receptor and calmodulin. Calmodulin enhances insulin-mediated receptor kinase activity and insulin stimulates phosphorylation of calmodulin.

Despite intensive research efforts, the functional role and regulation of the insulin receptor kinase remain enigmatic. In this investigation, we demonstrate that calmodulin enhances insulin-stimulated phosphorylation of the beta subunit of the insulin receptor and histone H2b and that insulin also stimulates phosphorylation of calmodulin. Using wheat germ lectin-enriched insulin receptor preparations obtained from rat adipocyte plasma membranes, calmodulin stimulated the rate and increased the amount of 32P incorporated predominantly into tyrosine residues of the beta subunit of the receptor when assayed in the presence of insulin. The stimulatory effect of calmodulin was both dose-dependent and saturable with half-maximal and maximal phosphorylation of the beta subunit occurring at 0.4 and 2.0 microM calmodulin, respectively. Ca2+ enhanced the ability of calmodulin to stimulate insulin-mediated phosphorylation of the beta subunit with an apparent K0.5 of approximately 0.6 microM. Calmodulin also induced an approximately 2-fold increase in both the rate and amount of insulin-mediated incorporation of 32P into histone H2b. The stimulatory effect of calmodulin was only observed in the presence of insulin and was concentration-dependent (K0.5 approximately 3.0 microM calmodulin), saturable (at 5 microM calmodulin), and Ca2+-dependent (K0.5 = 0.2 microM free Ca2+). Insulin also induced phosphorylation of a 17-kDa protein. On the basis of its molecular weight and purification via immunoadsorption with protein A-Sepharose-bound anti-calmodulin IgG, this phosphoprotein was identified as a phosphorylated form of calmodulin. Phosphorylation of calmodulin was only observed in the presence of insulin and was both Ca2+- and insulin concentration-dependent with half-maximal effects observed at 0.1 microM free Ca2+ and 350 microunits/ml insulin. Collectively, these results support the hypothesis that Ca2+ and calmodulin participate in the molecular mechanism whereby binding of insulin to its receptor is coupled to changes in cellular metabolism.

A calmodulin dependent Ca2+-activated K+ channel in the adipocyte plasma membrane.

Increased membrane permeability (conductance) that is specific for K+ and directly activated by Ca2+ ions, has been identified in isolated adipocyte plasma membranes using the K+ analogue, 86Rb+. Activation of these K+ conductance pathways (channels) by free Ca2+ was concentration dependent with a half-maximal effect occurring at 32 +/- 4 nM free Ca2+ (n = 7). Addition of calmodulin further enhanced the Ca2+ activating effect on 86Rb+ uptake (K+ channel activity). Ca2+-dependent 86Rb+ uptake was inhibited by tetraethylammonium ion and low pH. It is concluded that the adipocyte plasma membrane possesses K+ channels that are activated by Ca2+ and amplified by calmodulin.