Abstract
A phosphatidylinositol 4-kinase activator (PIK-A49) has been purified from carrot cells grown in suspension culture. The activator was purified from a soluble fraction using DEAE-Sepharose CL-6B and S-Sepharose chromatography columns. PIK-A49 has a relative molecular mass of 49 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The A50 for the activation of the Triton X-100-solubilized phosphatidylinositol 4-kinase fraction was 0.1 microM. Maximal activation was 3-4-fold. The analysis of the sequences of seven peptide fragments containing a total of 142 amino acid residues indicated that PIK-A49 was 69% identical to an actin-binding protein (ABP-50) from Dictyostelium and > 90% identical to elongation factor-1 alpha (EF-1 alpha) from carrot, tomato, and Arabidopsis. PIK-A49 bound actin and facilitated actin polymerization. Poly(U)-directed polyphenylalanine synthesis assays indicated that PIK-A49 had EF-1 alpha activity. The EF-1 alpha activity was enhanced by rabbit EF-1 beta gamma. Activation of phosphatidylinositol 4-kinase by a protein that binds actin and that has EF-1 alpha activity provides additional complexity to the signal transduction mechanisms involving inositol phospholipid metabolism.
Highlights
PI 4-kinase has been shown to be activated by positively charged compounds or polypeptides and inhibited by quercetinand heparin(Carpenterand Cantley, 1990;Vogel and EF-1&
The actin-bound PI 4-kinase P serves as theprecursor of phosphatidylinositol 4,5-bisphos- activity is responsive to in vivo treatments with epidermal phate, which is the source of the second messengers inositol growth factor in A431 cells (Payrastre et al, 1991) and with
phosphatidylinositol 4-monophosphate (PI-4-P) caninhibit the carrot cells, we previously found that exogenous phospholiactivity of a synaptosomal protein kinase (Sahyoun et al, pase Az released a plasma membrane PI 4-kinase that could 1989) and can stimulate the activity of the Na+-K+-ATPase be activated by a soluble, heat-stable, and trypsin-sensitive (Palatini et al, 1977),the Ca2+-ATPase(Missiaen et al, 1989) component that had a relative molecular mass of70 kDa (Gross et al, 1992)
Summary
The abbreviations used are: PI, phosphatidylinositol; EF, elongation factor; PAGE, polyacrylamidegel electrophoresis; PI-4-P, Materials-Wild carrot (Daucus carota L.) cells were originally derived from embryogenic cultures (Boss et al, 1984). Thecells were phosphatidylinositol 4-monophosphate; HPLC, high pressure liquid chromatography. M. H.Cho and W .F. Boss, unpublisheddata. Grown in suspension culture, transferred weekly, and used on the 4th day aftertransfer as previously described (Chen and Boss, 1990)
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