Immunocytochemical Detection of Phosphatidylinositol 3 Kinase in Burkitt Lymphoma Cells.

Abstract

ABSTRACT. PI 3-kinase, an enzyme responsible for the phosphorylation of the D3 position of the inositol ring of phosphatidylinositol (PI), is recognized to be involved in the regulation of many cellular processes such as mitogenic signalling, inhibition of apoptosis, intracellular vesicle trafficking/secretion, regulation of actin and integrin functions and regulation of protein kinases induced by tumour necrosis factor, oncoproteins and ultraviolet light. Here we report the subcellular distribution and the phosphorylative pattern of p85 a subunit of PI 3-kinase in Burkitt lymphomacells exposed to R interferon a treatment. Immunocytochemical analysis of this enzyme, performed by con focal microscopy, revealed an increased expression of this protein at cytoplasmic level after 90 min of interferon a treatment. Western blotting analyses performed on nuclear and cytoplasmic fractions confirmed the overexpression found by con focal microscopy at cytoplasmic level in the 90 min interferon a treated cells still persisting in the 24 hr treated samples. Such an overexpression was paralleled by an increase of tyrosine phosphorylation both at cytoplasmic and nuclear level suggesting that an enhanced requirement for cytoplasmic expression and phosphorylation of PI 3-kinase might be necessary to the cell for regulating some cytoplasmic-nuclear cross talk involved in the control of Burkitt lymphomacell metabolism following interferon a treatment.Amongthe pathways activated by different exogenous stimuli at plasma membrane level inositol lipids are known to play a pivotal role (2, 12, 23). Such a signalling system was found also involved in the downstream metabolic events related to the growth inhibitory action of R interferon a in Daudi lymphomacells (5-9). Indeed, results on the expression and the activity of different isoforms of phosphatidylinositol phospholipase C (PI-PLC), the enzyme responsible for the hydrolysis of phosphatidylinositolbisphosphate (PIP2) producing the well known second messengers diacylglycerol (DAG) and inositoltriphosphate (IP3), evidenced a peculiar involvement of different PLCisoforms in the cytoplasmic and nuclear compartments in response to interferon treatment (10-1 1). Moreover, amongthe enzymescorrelated to the inositol lipid metabolism activated by cytokine receptor tyrosine kinase, recently phosphatidylinositol 3-kinase (PI 3-kinase), which phosphorylates the D3 position of the inositol ring of phosphatidylinositol (PI) to produce PI3P, PI3,4P2 and PI3,4,5P3, received particular attention as possible regulator of many cellular processes (16). PI3,4P2 and PI3,4,5P3, are not found in the normal phosphoinositide cycle and do not appear to be substrate for phosphatidylinositol specific phospholipase C, acting themselves directly as second messengers (21).PI 3-kinase is a dimeric enzyme characterized by a catalytic pi 10 subunit and a regulatory p85 subunit. The latter lacks catalytic activity, works as an adapter protein with one SH3and two SH2 domains and associates with and serves as substrate...