Genetic and functional studies of phosphatidyl-inositol 4-kinase type IIIα

Abstract

Phosphatidylinositol 4-kinase type IIIa (PI4KIIIα) is one of four mammalian PI 4-kinases that catalyzes the first committed step in polyphosphoinositide synthesis. PI4KIIIα has been linked to regulation of ER exit sites and to the synthesis of plasma membrane phosphoinositides and recent studies have also revealed its importance in replication of the Hepatitis C virus in liver. Two isoforms of the mammalian PI4KIIIα have been described and annotated in GenBank: a larger, ~ 230 kDa (isoform 2) and a shorter splice variant containing only the ~97 kDa C-terminus that includes the catalytic domain (isoform 1). However, Northern analysis of human tissues and cancer cells showed only a single transcript of ~ 7.5 kb with the exception of the proerythroleukemia line K562, which contained significantly higher level of the 7.5 kb transcript along with smaller ones of 2.4, 3.5 and 4.2 kb size. Bioinformatic analysis also confirmed the high copy number of PI4KIIIα transcript in K562 cells along with several genes located in the same region in Chr22, including two pseudogenes that cover most exons coding for isoform 1, consistent with chromosome amplification. A panel of polyclonal antibodies raised against peptides within the C-terminal half of PI4KIIIα failed to detect the shorter isoform 1 either in COS-7 cells or K562 cells. Moreover, expression of a cDNA encoding isoform 1 yielded a protein of ~97 kDa that showed no catalytic activity and failed to rescue hepatitis C virus replication. These data draw attention to PI4KIIIα as one of the genes found in Chr22q11, a region affected by chromosomal instability, but do not substantiate the existence of a functionally relevant short form of PI4KIIIα.

Mapping of Functional Domains of the Lipid Kinase Phosphatidylinositol 4-Kinase Type III Alpha Involved in Enzymatic Activity and Hepatitis C Virus Replication

“…Therefore, we generated a panel of PI4KIII␣ deletion mutants and characterized them regarding their enzymatic activity, their ability to be activated by expression of the HCV polyprotein, their efficiency in rescuing HCV replication, their subcellular localization in the presence and absence of HCV proteins, and their interaction with HCV NS5A. We found a very close linkage between enzymatic activity, induction of PI4P, and rescue of HCV replication, in line with our previous results and those of others, suggesting that the catalytically active center of PI4KIII␣ is essential for its role in HCV replication (10,15). These data were largely connected to the ability of PI4KIII␣ to be recruited to the HCV replication sites.…”

“…Earlier studies identified an enzymatically active mutant which lacked the N-terminal 872 amino acids, while deletion of 1,189 amino acids entirely abrogated enzymatic function (corresponding to residues 931 and 1249, respectively, in our PI4KIII␣ gene), arguing for a 130-kDa enzyme as a minimal requirement for enzymatic activity (20,52,53), whereas an alternative splice variant of PI4KIII␣ lacking 1,249 N-terminal amino acids was shown to be inactive and did not support HCV replication (15). Our deletion analysis now revealed that the N-terminal 1,152 amino acids are dispensable for enzymatic activity, whereas further deletion of 50 amino acids resulted in an inactive protein (⌬1-1202).…”

“…Interestingly, in cDNA libraries of various tissues and cell lines, an alternative splice variant of PI4KIII␣ was discovered that lacked the coding sequence for the N-terminal 1,249 amino acids of the 240-kDa variant. This truncated variant was found to be catalytically inactive in vitro and did not support HCV replication, pointing to an important role of residues further upstream in enzymatic function (15) but questioning the general significance of this isoform.…”

Phosphatidylinositol 4-kinases: hostages harnessed to build panviral replication platforms

“…These data indicate that PtdIns4P lipids are required for HCV replication and are an important clinical hallmark of the disease. The existence of isoform 1 at the protein level is questionable; if it exists, it is a catalytically inactive protein [91]. b EGF: epidermal growth factor; GGA: GGA: Golgi-localized, gamma adaptin ear-containing, ARF-binding protein; PM: plasma membrane.…”

Evaluation of Phosphatidylinositol-4-Kinase IIIα as a Hepatitis C Virus Drug Target

“…PI4KIII␣ is one of four mammalian phosphatidylinositol 4-kinases that catalyze the first step in phosphoinositide synthesis (5). PI4KIII␣ is a 230-kDa protein (58) that is primarily localized in the endoplasmic reticulum (67) and apparently contributes to the formation of endoplasmic reticulum exit sites (14,24) as well as the maintenance of plasma membrane phosphoinositide pools (6).…”