Synthase Kinase-3α Research Articles

Selective Interaction of Protein Kinase FA/Glycogen Synthase Kinase-3α with Membrane Phospholipids

Previously we reported that the activity of protein kinase FA/glycogen synthase kinase-3α (kinase FA/GSK-3α) can be detected in several brain membrane fractions. In this report, we examined whether kinase FA/GSK-3α can directly interact with membrane phospholipids by using anti-kinase FA/GSK-3α antibody as a more specific studying tool. It was found that kinase FA/GSK-3α can associate with NaOH-extracted brain membranes and selectively interact with several kinds of reconstituted phospholipid vesicles including phosphatidic acid (PA), phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI), and phosphatidyl serine (PS) vesicles. Increasing ionic strength in the reaction could disrupt the interaction between kinase FA/GSK-3α and PA, PI, or PE vesicles but had no effect on the interaction between kinase FA/GSK-3α and PS vesicles, indicating that both ionic and non-ionic interactions are involved in this process, respectively. Moreover, both kinase activity and protease sensitivity of kinase FA/GSK-3α can be affected profoundly by these phospholipid vesicles and different forms of the kinase can be produced when it binds to distinct types of phospholipid vesicles. Taken together, the results demonstrate a direct interaction of kinase FA/GSK-3α with membrane phospholipids and suggest that membrane phospholipids may be directly involved in regulating kinase FA/GSK-3α activity.

Reversible tyrosine phosphorylation/dephosphorylation of proline-directed protein kinase FA/glycogen synthase kinase-3α in A431 cells

Modulation of protein kinase FA/glycogen synthase kinase-3alpha (kinase FA/GSK-3alpha) by reversible tyrosine phosphorylation/dephosphorylation was investigated. In addition to genistein, other protein tyrosine kinase (PTK) inhibitors, such as tyrphostin A47 and B42, also could induce tyrosine dephosphorylation and inactivation of kinase FA/GSK-3alpha in A431 cells, and this process was found to be reversible. Pretreatment of the cells with 100 microM orthovanadate, a protein tyrosine phosphatase (PTP) inhibitor, could diminish significantly the effects of PTK inhibitors on both enzyme activity and phosphotyrosine content of the kinase, suggesting that the PTK inhibitors induced tyrosine dephosphorylation/inactivation of this kinase is mediated by orthovanadate-sensitive PTP(s) in A431 cells. Moreover, the phosphotyrosine moiety of kinase FA/GSK-3alpha was found to be highly turned over in resting cells. Interestingly, we found that the less active, tyrosine-dephosphorylated form of kinase FA/GSK-3alpha immunoprecipitated from genistein-treated cells was able to reactivate partially with concomitant rephosphorylation of tyrosine residue in vitro. Taken together, these findings demonstrate that tyrosine phosphorylation and concomitant activation of kinase FA/GSK-3alpha can be carried out both in vitro and in vivo and an in vivo phosphatase activity may function in antagonism to PTK activation of kinase FA/GSK-3alpha.

Endothelin-1 and Insulin Induce Cellular Inactivation of Protein Kinase F(A)/Glycogen Synthase Kinase-3alpha in a Common Signaling Pathway.

In this study, we investigate the effects of endothelin-1 (ET-1) and insulin on the cellular activity of protein kinase F(A)/glycogen synthase kinase-3alpha (kinase F(A)/GSK-3alpha) in rat adipocytes. The cellular activity of kinase F(A)/GSK-3alpha is inhibited to approximately 50% of control within 30 min when cells are treated with 1 nM ET-1 at 37 degrees C; in addition, significant inhibition to approximately 60% of control is observed at as low as 1 pM ET-1. Conversely, ET-1 at concentrations up to 1 nM has no direct effect on purified kinase F(A)/GSK-3alpha in vitro. Immunoblotting analysis further reveals that the protein level of this kinase is not significantly changed when treated with 1 nM ET-1 for 30 min. Similar to ET-1, insulin as low as 10 nM can also induce inactivation of kinase F(A)/GSK-3alpha to approximately 50% of control in adipocytes when processed under identical conditions. Most importantly, when treated with both insulin and ET-1, the activity of kinase F(A)/GSK-3alpha can be decreased only to approximately 50% of control. Taken together, the results provide initial evidence that ET-1 and insulin may regulate this important multisubstrate/multifunctional protein kinase in a common signaling pathway in cells. Copyright 1996 S. Karger AG, Basel