Science 2000 Mar; 287:2026-29

Authors: Odom AR, Stahlberg A, Wente SR, York JD.
 

Presenter:  Christine Misquitta

Background

 The enzyme, phospholipase C, is most well known for its role in the intracellular
induction of inositol phospholipids.  The most important of these being two phosphorylated
derivatives of PI, PI-phosphate (PIP) and PI-bisphosphate (PIP2).  Many intracellular events are
linked to the signalling molecules, IP3 and DAG, formed from the breakdown of PIP2.  While IP3
is central to calcium release from internal stores, its further derivatives, IP4, IP5, IP6 may also be
important in other intracellular signals.

 One additional role for IP products may be in gene expression through control of
transcription.  In Figure 1A, below, the ArgR-Mcm1 protein complex is shown to control
transcription1 .  The proteins Arg80, Arg81, Arg82 and Mcm1 make up the ArgR-Mcm1
complex.  The first two proteins are arginine-specific in their transcriptional control while the
latter two are non-specific regulators affecting processes from mating, cell-cycle control and
nutrient responses.  The link of  PLC and IP signalling to transcriptional control is via Arg82
which is a dual-specificity IP3-IP4 kinase.

Figure 1.

Summary of present work

The key experiments, with their purpose and results, are summarized below.

1) PURPOSE: to test directly whether ARG82 encodes an intrinsic IP3 kinase
RESULT: purified, recombinant Arg82 included with [3H]inositol 1,4,5-trisphosphate and ATP
showed that Arg82 performs 2 phosphorylation steps
CONCLUSION: Arg82 is a dual-specificity IP3 6-kinase or IP4 3-kinase

The authors then designate Arg82 as Ipk2 (inositol polyphosphate kinase)

2) PURPOSE: to test the functional role of Ipk2 in vivo
RESULT: using ipk2 mutant yeast strains, cells lacking Ipk2 showed a loss of  IP4, IP5, and IP6
but had increased amounts of IP3 and an IP2x (an unidentified IP2 isomer)

3) PURPOSE: to examine the relationship between PLC and Ipk2
RESULTS:
a) in wild-type cells, overexpression of PLC resulted in large increases of IP3, IP4, IP5, and IP6
levels
b) in ipk2? cells, overexpression of PLC increased levels of IP3 and IP2x 20-fold while IP4, IP5,
and IP6 were undetectable
CONCLUSION: PLC activity produces the IP3 substrate for Ipk2 in vivo

In previous studies a role for Arg82 (Ipk2) in gene transcription regulation was determined.
through the protein complex ArgR-Mcm1 as depicted in Figure 1A.

4) PURPOSE: characterize the role of Ipk2-mediated IP production in the formation of
ArgR-Mcm1 protein-DNA complexes
RESULTS:
a) a band of large molecular size was seen with wild-type extracts in gel shift assays
corresponding to a multi-protein complex
b) when extracts were taken from ipk2? cells, no shift was detected
c) reintroduction of Ipk2 into the null strain restored the band shift
 

Why this paper is important

 This paper shows the direct control of the production of downstream IP products
(specifically, production of IP4 and possibly IP5, but not IP6) by Ipk2 is necessary for
ArgR-Mcm1-mediated gene expression.   In addition, Gle1-mediated mRNA export is modulated
by IP6, whose production requires phosphorylation of the D-2 position of IP5 by Ipk1 (Figure 1B).
The authors state that control of gene expression through Ipk2 and regulation of mRNA export
through Ipk1 may contribute to the mechanisms by which diverse cellular agonists induce
selective responses.
 By showing that Arg82 (Ipk2) of the ArgR-Mcm1 protein complex is dependent on PLC,
their work here also uncovers some other important roles that may exist for PLC in addition to
calcium signalling within the cell.

Author’s Abstract:

 Phospholipase C and two inositol polyphosphate (IP) kinases constitute a signalling
pathway that regulates nuclear messenger RNA export through production of inositol
hexakisphosphate (IP6). The inositol 1,4,5-trisphosphate kinase of this pathway in
Saccharomyces cerevisiae, designated Ipk2, was found to be identical to Arg82, a regulator of the
transcriptional complex ArgR-Mcm1. Synthesis of inositol 1,4,5,6-tetrakisphosphate, but not IP6,
was required for gene regulation through ArgR-Mcm1. Thus, the phospholipase C pathway
produces multiple IP messengers that modulate distinct nuclear processes. The results reveal a
direct mechanism by which activation of IP signalling may control gene expression.
 

References:

1.  www.sciencemag.org/feature/data/1045815.shl