Polylysine activates and alters the divalent cation requirements of the insulin receptor protein tyrosine kinase

Rosen, Ora M.; Lebwohl, David

doi:10.1016/0014-5793(88)80858-5

Cited by 44 publications

(15 citation statements)

References 12 publications

(4 reference statements)

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“…S6). The activation of insulin signaling by PLL is consistent with the results of previous studies that PLL activates insulin receptor protein kinase by interacting with its β-subunit (Fujita-Yamaguchi et al 1989 ; Rosen and Lebwohl 1988 ). Furthermore, PLL increased the levels of pAkt and pERK compared to treatment with insulin alone when preadipocytes were treated with insulin and PLL at the same time (Fig.…”

Section: Discussionsupporting

confidence: 91%

α-Poly-l-lysine functions as an adipogenic inducer in 3T3-L1 preadipocytes

Lee

et al. 2021

Amino Acids

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Abstractα-Poly-l-lysine (PLL) has been used for various purposes such as cell attachment, immunization, and molecular delivery, and is known to be cytotoxic to several cell lines. Here, we studied the effect of PLL on the adipogenesis of 3T3-L1 cells and investigated the underlying mechanism. Differentiation media containing PLL with a molecular weight (MW) greater than 4 kDa enhanced lipid droplet formation and increased adipogenic marker levels, indicating an increase in adipocyte differentiation. PLL with a molecular weight between 30 and 70 kDa was more effective than PLL of other sizes in 3T3-L1 cell differentiation. Moreover, PLL induced 3T3-L1 adipogenesis in insulin-free adipocyte differentiation medium. Incubation with insulin and PLL exhibited greater adipogenesis than insulin treatment only even at a high concentration. PLL stimulated insulin signaling and augmented the signaling pathway when it was added with insulin. While PLL did not activate the glucocorticoid receptor, which is phosphorylated by dexamethasone (DEX), it showed a positive effect on the cAMP signal pathway when preadipocytes were treated with PLL and 3-isobutyl-1-methylxanthine (IBMX). Consistent with these results, incubation with PLL and DEX without IBMX induced adipocyte differentiation. We also observed that the mitotic clonal expansion phase was the critical stage in adipogenesis for inducing the effects of PLL. These results suggest that PLL functions as an adipogenic inducer in 3T3-L1 preadipocytes and PLL has a direct effect on insulin signaling, one of the main regulatory pathways.

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Section: Discussionsupporting

confidence: 91%

α-Poly-l-lysine functions as an adipogenic inducer in 3T3-L1 preadipocytes

Lee

et al. 2021

Amino Acids

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“…Cyclic PIP biosynthesis has been described in purified plasma membranes. There may be reservation that activation of cyclic PIP synthase by protamine would be related to activation of a tyrosine kinase, however, it appears most likely that two drugs, protamine and biguanide, which are both shown to activate insulin receptor tyrosine kinase (Rosen and Lebwohl, 1988;Dominguez et al, 1996), would also activate cyclic PIP synthase by tyrosine phosphorylation. The activation of cyclic PIP synthase by fluoride (10 mM) or by GMP-PNP (Figure 3) should be related to activation of the enzyme by G proteins and activation by protamine or biguanides should be related to activation by insulin, as these two compounds are reported to activate the insulin receptor tyrosine kinase (Rosen and Lebwohl, 1988;Dominguez et al, 1996).…”

Section: Discussionmentioning

confidence: 99%

“…Figure 3 shows the concentration-dependent activation of cyclic PIP synthase by the stable GTP-analog GMP-PNP (guanylylimidodiphosphate), a G protein activator (Pfeuffer and Helmreich, 1975). Protamine (Rosen and Lebwohl, 1988) and biguanide (Dominguez et al, 1996) are both known to activate the insulin receptor tyrosine kinase. Furthermore, addition of protamine or biguanide to assays for cyclic PIP synthase activity results in a 4-fold activation of the enzyme, which is additive with respect to the activation by fluoride (10 mM).…”

Section: Kinetic Parameters Of Cyclic Pip Synthasementioning

confidence: 99%

Two Different Mechanisms for Activation of Cyclic PIP Synthase: by a G Protein or by Protein Tyrosine Phosphorylation

Wasner¹,

Gebel²,

Hucken³

et al. 2000

Biological Chemistry

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The biosynthesis of the functional, endogenous cyclic AMP antagonist, prostaglandylinositol cyclic phosphate (cyclic PIP) is performed by the plasma membrane-bound enzyme cyclic PIP synthase, which combines prostaglandin E (PGE) and activated inositol phosphate (n-IP) to cyclic PIP. The Km values of the enzyme for the substrates PGE and n-IP are in the micromolar range. The plasma membrane-bound synthase is activated by fluoride, by the stable GTP analog GMP-PNP, by protamine or biguanide, by noradrenaline, and by insulin. The activation by protamine or biguanide and fluoride (10 mM) is additive, which may indicate the presence of two different types of enzyme, comparable to phospholipase Cbeta and phospholipase Cgamma. Plasma membrane-bound cyclic PIP synthase is inhibited by the protein tyrosine kinase inhibitor tyrphostin B46 with an IC50 of 1.7 microM. However, the solubilized and gel-filtrated enzyme is no longer inhibited by tyrphostin, indicating that the activity of cyclic PIP synthase is connected with the activity of a membrane-bound protein tyrosine kinase. Cyclic PIP synthase activity of freshly prepared plasma membranes is unstable. Upon freezing and rethawing of liver plasma membranes, this instability is increased about 2-fold. Protein tyrosine phosphatase inhibitors [vanadate, fluoride (50-100 mM)] stabilize the enzyme activity, but protease inhibitors do not, indicating that inactivation of the enzyme is connected with protein tyrosine dephosphorylation. Cyclic PIP synthase is present in all tissues tested, like brain, heart, intestine, kidney, liver, lung, skeletal muscle, spleen, and testis. Apart from liver, cyclic PIP synthase activity in most tissues is rather low, but it can be increased up to 5-fold when protein tyrosine phosphatase inhibitors like vanadate are present in the homogenization buffer. Preincubation of cyclic PIP synthase of liver plasma membranes with the tyrosine kinase src kinase causes a 2-fold increase of cyclic PIP synthase activity, though this is certainly not the physiological role played by src kinase in intact cells. The data indicate that cyclic PIP synthase can be activated by two separate mechanisms: by a G protein or by protein tyrosine phosphorylation.

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“…Previously, we (11) and others (8,(15)(16)(17) have reported that addition of polycations (such as protamine and poly-L-lysine) to the phosphorylation reaction greatly enhances both the initial velocity of the enzyme and the maximal phosphate incorporation into the soluble insulin receptor kinase domain. It has been demonstrated that the effect of poly-L-lysine is due to a nonspecific kinase aggregation, presumably leading to a more efficient transphosphorylation of the kinases (17).…”

Section: Construction and Purification Of Monomeric And Dimericmentioning

confidence: 98%

Dimerization-Induced Activation of Soluble Insulin/IGF-1 Receptor Kinases: An Alternative Mechanism of Activation

et al. 2001

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To study the role of kinase dimerization in the activation of the insulin receptor (IR) and the insulin-like growth factor receptor-1 (IGF-1R), we have cloned, expressed, and purified monomeric and dimeric forms of the corresponding soluble kinase domains via the baculovirus expression system. Dimerization of the kinases was achieved by fusion of the kinase domains to the homodimeric glutathione S-transferase (GST). Kinetic analyses revealed that kinase dimerization results in substantial increases (10-100-fold) in the phosphotransferase activity in both the auto- and substrate phosphorylation reactions. Furthermore, kinase dimerization rendered the autophosphorylation reaction concentration-independent. However, whereas dimerization was required for the rapid autophosphorylation of the kinases, it was not essential for the enhanced kinase activity in substrate phosphorylation reactions. Comparison of HPLC-phosphopeptide maps of the monomeric and dimeric kinases revealed that dimerization leads to an increased phosphorylation of the regulatory activation loop of the kinases, strongly suggesting that bis- and trisphosphorylation of the activation loop are mediated by transphosphorylation within the kinase dimers. Most strikingly, limited proteolysis revealed that GST-mediated dimerization by itself had a major impact on the conformation of the activation loop by stabilizing a conformation that corresponds to the active, phosphorylated form of the kinase. Thus, in analogy to the insulin/IGF-1-ligated holoreceptors, the dimeric GST-kinases are primed to rapid autophosphorylation by an increase in the local concentration of both phosphoryl donor and phosphoryl acceptor sites and by a dimerization-induced conformational change of the activation loop that leads to an efficient transphosphorylation of the regulatory tyrosine residues.

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Polylysine activates and alters the divalent cation requirements of the insulin receptor protein tyrosine kinase

Cited by 44 publications

References 12 publications

α-Poly-l-lysine functions as an adipogenic inducer in 3T3-L1 preadipocytes

α-Poly-l-lysine functions as an adipogenic inducer in 3T3-L1 preadipocytes

Two Different Mechanisms for Activation of Cyclic PIP Synthase: by a G Protein or by Protein Tyrosine Phosphorylation

Dimerization-Induced Activation of Soluble Insulin/IGF-1 Receptor Kinases: An Alternative Mechanism of Activation

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