The 1,4-naphthoquinone derivative, shikonin, has been shown to increase glucose uptake by adipocytes and myocytes with minor effects on protein tyrosine phosphorylation in the cells (Biochem Biophys Res Commun 292:642-651, 2002). The present study was performed to examine the mechanism of this action of shikonin. Shikonin inhibited the phosphatidylinositol 3,4,5-triphosphate (PtdIns-3,4,5-P 3 ) phosphatase activity of recombinant phosphatase and tensin homolog deleted on chromosome 10 (PTEN) with an IC 50 value of 2.7 M. Shikonin induced marked accumulation of PtdIns-3,4,5-P 3 and activation of protein kinase B (PKB) in Chinese hamster ovary cells expressing insulin receptors. In addition to its effect on PTEN, shikonin was found to inhibit several protein phosphatases in cell-free systems. Its effect on tyrosine phosphorylation in intact cells was far weaker than that of pervanadate, a widely used tyrosine phosphatase inhibitor, despite the observation that the effect of shikonin on PKB was more potent than that of pervanadate. These results suggested that the inhibition of PTEN provides a clue to its potent insulin-like actions. We also found that naphthoquinones, including 1,2-naphthoquinone, inhibit PTEN in the cell-free system, which suggested that the effect on PTEN (and thus the effect on phosphatidylinositol 3-kinase signaling) should be taken into account when examining the pharmacological actions of naphthoquinone derivatives.Binding of insulin to its specific receptors causes tyrosine phosphorylation and activation of the receptors, which in turn induces the activation of downstream signaling molecules. Phosphoinositide 3-kinase (PI 3-kinase) is one of the factors involved in transduction of the insulin signal from the receptors (Wymann and Pirola, 1998). The SH2 domains of the regulatory subunit of PI 3-kinase bind to the phosphorylated tyrosine residues in the consensus sequence, YXXM. This binding activates PI 3-kinase and results in the accumulation of PtdIns-3,4,5-P 3 and PtdIns-3,4-P 2 . The machinery downstream of PI 3-kinase includes a serine/threonine kinase protein kinase B (PKB, also known as Akt), the activity of which is up-regulated by the products of PI 3-kinase (Alessi and Downes, 1998). Overexpression of a constitutively active form of PKB has been reported to lead to increased glucose uptake and GLUT4 translocation in 3T3L1 adipocytes (Kohn et al., 1996).The tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) is an enzyme that dephosphorylates the D3 position of PtdIns-3,4,5-P 3 and PtdIns-3,4-P 2 (Maehama and Dixon, 1998;Cantley and Neel, 1999). Overexpression of the lipid phosphatase reduces the insulininduced accumulation of PtdIns-3,4,5-P 3 in 293 cells (Maehama and Dixon, 1998). Infection with adenovirus expressing PTEN in 3T3L1 adipocytes attenuates both the insulin-induced glucose uptake and the Akt activation (Nakashima et al., 2000). Microinjection of an anti-PTEN antibody potentiates the insulin-induced events (Ono et al., 2001). Thus, PT...

Menadione (vitamin K(3)) has been shown to activate Erk in several cell lines. This effect has been shown to be due to the activation of EGF receptors (EGFR) as a result of inhibition of some protein tyrosine phosphatases. In the present study, we examined the effects of menadione on Akt in Chinese hamster ovary cells. The phosphorylation of Akt by menadione was not inhibited by AG1478, an inhibitor of EGFR. Menadione inhibited the lipid phosphatase activity of PTEN in a cell-free system. In an intact cell system, menadione inhibited the effect of transfected PTEN on Akt. Thus, one mechanism of its action was considered the accelerated activation of Akt through inhibition of PTEN. This was not the sole mechanism responsible for the EGFR-independent activation of Akt, because menadione attenuated the rate of Akt dephosphorylation even in PTEN-null PC3 cells. The decelerated inactivation of Akt, probably through inhibition of some tyrosine phosphatases, was considered another mechanism of its action.

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Tektins (TEKTs) are filamentous proteins associated with microtubules in cilia, flagella, basal bodies, and centrioles. Five TEKTs (TEKT1, -2, -3, -4, and -5) have been identified as components of mammalian sperm flagella. We previously reported that TKET1 and -3 are also present in the heads of rodent spermatozoa. The present study clearly demonstrates that TEKT2 is present at the acrosome cap whereas TEKT3 resides just beneath the plasma membrane of the post-acrosomal region of sperm heads in unactivated bull spermatozoa, and builds on the distributional differences of TEKT1, -2, and -3 on sperm heads. We also discovered that hyperactivation of bull spermatozoa by cell-permeable cAMP and calyculin A, a protein phosphatase inhibitor, promoted translocation of TEKT3 from the post-acrosomal region to the equatorial segment in sperm heads, and that TEKT3 accumulated at the equatorial segment is lost upon acrosome reaction. Thus, translocation of TEKT3 to the equatorial segment may be a capacitation- or hyperactivation-associated phenomenon in bull spermatozoa. Mol. Reprod. Dev. 84: 30-43, 2017. © 2016 Wiley Periodicals, Inc.

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