The Journal of Immunology, Vol 137, Issue 3 971-976, Copyright © 1986 by American Association of Immunologists

ARTICLES

Identification and characterization of the phosphatidylinositol kinase in membranes of murine T lymphocytes

JJ O'Shea, JB Harford and RD Klausner

Polyphosphoinositide metabolism appears to be a widely employed mechanism by which membrane receptors transduce the signal to activate cells. This pathway has been implicated in the activation of T lymphocytes. Unlike many receptor systems in which decreased levels of polyphosphoinositides are observed, T lymphocytes demonstrate increased concentrations of polyphosphoinositides as well as phosphoinositols (particularly IP3) on activation by mitogens. To understand the early biochemical events involved in T cell activation, we sought to identify and characterize the enzyme responsible for phosphorylating phosphatidylinositol (PI) to phosphatidylinositol 4-phosphate (PIP) in murine T lymphocytes. This kinase was found to be an integral membrane protein of T lymphocytes. It was found to be Mg2+ (apparent Km = 5 mM)- or Mn2+-dependent, whereas Ca2+ was noted to be a competitive inhibitor of Mg2+. ATP is the preferred substrate over GTP (apparent Km = 0.14 mM and 0.62 mM, respectively). The kinase is inhibited by both PIP and PI 4,5-bisphosphate (PIP2), but not by other phospholipids or angiotensin II (a substrate for tyrosine kinase). The enzyme activity has identical characteristics in membranes derived from a T cell hybridoma, thymocytes, and T cell clones. The enzyme migrated predominantly as a single peak with an apparent m.w. of approximately 60,000 on gel filtration chromatography. The relationship of this enzyme to viral oncogene products such as pp60v-src and p68v-ros is discussed.

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