Abstract
Calmodulin and phosphatidylinositol 3-kinase are vital components of a number of common intracellular events. Calmodulin, a ubiquitous Ca2+-dependent effector protein, regulates multiple processes in eukaryotic cells, including cytoskeletal organization, vesicular trafficking, and mitogenesis. Phosphatidylinositol 3-kinase participates in events downstream of the receptors for insulin and other growth factors. Here we demonstrate by coimmunoprecipitation and affinity chromatography that Ca2+/calmodulin associates with Src homology 2 domains in the 85-kDa regulatory subunit of phosphatidylinositol 3-kinase, thereby significantly enhancing phosphatidylinositol 3-kinase activity in vitro and in intact cells. Furthermore, CGS9343B, a calmodulin antagonist, inhibited basal and Ca2+-stimulated phosphorylation of phosphatidylinositol in intact cells. These data demonstrate a novel mechanism for modulating phosphatidylinositol 3-kinase and provide a direct link between components of two fundamental signaling pathways.
Highlights
Calmodulin and phosphatidylinositol 3-kinase are vital components of a number of common intracellular events
Alterations in levels of intracellular free Ca2ϩ ([Ca2ϩ]i)1 mediate the action of several hormones through the regulation of key enzymes; these signaling events are often orchestrated by the Ca2ϩ effector, calmodulin (CaM) [1]
Activation of phosphatidylinositol 3-kinase (PI3-kinase) in many tissues and cell types is required for mitogenesis, neuronal differentiation, and enhanced glucose transport [2]
Summary
Calmodulin and phosphatidylinositol 3-kinase are vital components of a number of common intracellular events. The 85-kDa regulatory subunit of PI3-kinase (p85) was precipitated from lysates of baculovirus-infected Sf9 cells expressing p85 with CaM-Sepharose but not with Sepharose alone, demonstrating an interaction between these two proteins (Fig. 1A). To identify the CaM-binding region of p85, CaM-Sepharose was incubated with GST fusion proteins containing various regions of p85. Endogenous p85 from CHO cell lysates bound to CaM-Sepharose only in the presence of Ca2ϩ (Fig. 1C).
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