Biphasic regulation of type II phosphatidylinositol-4 kinase by sphingosine: cross talk between glycero- and sphingolipids in the kidney

Abstract

Phosphatidylinositol-4 kinase (PI-4K) is responsible for the generation of phosphatidylinositol-4 phosphate (PtdIns(4)P), a bioactive signaling molecule involved in several biological functions. In this study, we show that sphingosine modulates the activity of the PI-4K isoform associated with the basolateral membranes (BLM) from kidney proximal tubules. Immunoblotting with an anti-α subunit PI-4K polyclonal antibody revealed the presence of two bands of 57 and 62kDa in the BLM. BLM-PI-4K activity retains noteworthy biochemical properties; it is adenosine-sensitive, not altered by wortmanin, and significantly inhibited by Ca(2+) at the μM range. Together, these observations indicate the presence of a type II PI-4K. Endogenous phosphatidylinositol (PI) alone reaches PI-4K half-maximal activity, revealing that even slight modifications in PI levels at the membrane environment promote significant variations in BLM-associated-PI-4K activity. ATP-dependence assays suggested that the Mg.ATP(2-) complex is the true substrate of the enzyme and that free Mg(2+) is an essential cofactor. Another observation indicated that higher concentrations of free ATP are inhibitory. BLM-associated-PI-4K activity was ~3-fold stimulated in the presence of increasing concentration of sphingosine, while in concentrations higher than 0.4mM, in which S1P is pronouncedly formed, there was an inhibitory effect on PtdIns(4)P formation. We propose that a tightly coupled regulatory network involving phosphoinositides and sphingolipids participate in the regulation of key physiological processes in renal BLM carried out by PI-4K.

Keywords: BLM; BLM-PI4K; Basolateral plasma membrane; DAG; DGK; EGTA; HPLC; Hepes; Kidney; N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid; NaN(3); PA; PMSF; Phosphatidylinositol-4 kinase; PtdIns(4)(32)P; PtdIns(4)P; Signaling; Sphingosine kinase; TLC; [(32)P(i)]P(i); diacylglycerol; diacylglycerol kinase; ethylene glycol bis(β-amino-ethyl ether)N,N,N′,N′-tetracetic acid; high performance liquid chromatography; kidney basolateral membranes; phenylmethylsulfonyl fluoride; phosphatidic acid; phosphatidylinositol-4 kinase from kidney basolateral membrane; phosphatidylinositol-4-phosphate; radioactive orthophosphate; radioactive phosphatidylinositol-4-phosphate; sodium azide; thin-layer chromatography.