The massive accumulation of j#-carotene by the halotolerant micro alga Dunaliella bardawil, in response to high light intensity and several other environmental factors, has been studied so far under different sets of fixed conditions. To determine the kinetics and characteristics of the induction of

Annexin II is a Ca2+-, phospholipid-, and actin- binding protein that was implicated in the regulation of vesicular traffic and endosome fusion. It is a known substrate for protein kinases including the platelet-derived growth factor receptor, src protein-tyrosine kinase, and protein kinase C. In the present study we investigated the possible involvement of annexin II in insulin signal transduction. Phosphorylation of annexin II in response to insulin treatment of intact Chinese hamster ovary (CHO)-T cells was detected by 5 min and reached maximal levels after a 2-3-h incubation with the hormone. However, unlike other receptor substrates, annexin II failed to undergo insulin-induced Tyr phosphorylation under conditions where receptor internalization was inhibited. This was evident in CHO cells, overexpressing the insulin receptor, in which internalization was inhibited either by tyrosine kinase inhibitors or by lowering the temperature to 4 degrees C, and in CHO cells overexpressing various insulin receptor mutants in which normal internalization was impaired. Hence, Tyr phosphorylation of annexin II could be part of the internalization and sorting mechanism of the insulin receptor.

The effects of cationic polyamino acids on phosphorylation of the insulin and insulin-like growth factor 1 receptor kinases were studied and the following observations were made. (a) Polylysine stimulated both tyrosine and serine phosphorylation of the insulin receptor and of additional proteins present in lectin-purified membrane preparations from rat liver. (b) Polylysine synergized with insulin to enhance phosphorylation of the insulin receptor and of additional proteins (pp40 and ppl10). (c) Polylysine effects were more pronounced upon increasing the polylysine chain length. (d) The effect of polylysine was biphasic with an optimum at 100 pg/ml. (e) Polylysine was found ineffective in stimulating the phosphorylation of immobilized insulin receptors. Taken together, these findings support the notion that the action of polylysine involves conformational changes and presumably aggregation of soluble receptors. The same effects of polylysine were obtained with highly purified insulin receptor preparations. Under these conditions polylysine enhanced both serine and tyrosine phosphorylation of the insulin receptor, suggesting that polylysine stimulates the activity of the insulin receptor kinase, and of a serine kinase that is tightly associated with the insulin receptor.The response of cells to the polypeptide hormone insulin is initiated through binding of insulin to its cell-surface receptor. The receptor is a transmembrane glycoprotein consisting of two a-subunits (135 kDa) and two p-subunits (95 kDa), linked by disulfide bonds (for recent reviews see [I -31). The interaction of insulin with the receptor's a-subunits is transformed into a transmembrane message, presumably by receptor crosslinking or aggregation [4, 51, which results in conformational changes in the intracellular portion. In intact cells, the next step of insulin action involves phosphorylation of the insulin receptor P-subunit on serine, threonine and tyrosine residues, yet the purified solubilized receptor undergoes insulin-dependent phosphorylation exclusively on tyrosine residues. This reaction is catalyzed by the receptor P-subunit which functions as an insulin-dependent tyrosine kinase (IRK) capable of phosphorylating itself and exogenous substrates [l -31.Since the insulin receptor functions as a protein kinase, studies on the next step in insulin action are focused on intracellular proteins which are tyrosine phosphorylated in an insulin-dependent manner. Such proteins might serve as substrates for the activated insulin receptor. One of the problems in identifying substrates for the insulin receptor is their low degree of phosphorylation, which results either from their scarcity, high dephosphorylation rate or poor recovery through the purification stages and assay. One way to overcome this problem is to use agents that potentiate the autophosphorylation and activity of the IRK. Several basic proteins, such as protamine sulfate, histones and polylysine, were previously reported to stimulate in vitvo receptor autophosphorylation [6 -121 ...

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