Cell signaling by a physiologically reversible inositol phosphate kinase/phosphatase

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Introduction

Control over a physiological process can be tuned to an especially fine level by its regulation through a dynamic balance between signals with opposing influences. This is, for example, a situation that arises when two metabolic substrates are interconverted through the actions of competing enzymes. This bidirectional interconversion is known as a substrate cycle (Hers and Hue, 1983), although, in less enlightened times, it was often called a “futile” cycle. Another illustration of this phenomenon is provided by the manipulation of the phosphorylation status of a target protein by competing activities of protein kinases and protein phosphatases (Oliver and Shenolikar, 1998). A greatly amplified output signal can also result from quite delicate shifts in the balance of opposing stimulatory and inhibitory input signals can. This process—coincident integration of negative and positive signaling inputs—also bestows signaling specificity and regulates “cross-talk” between distinct signal transduction pathways. It is a major challenge to define these interactions at a molecular level. An even more complex task is to understanding the interactions between signaling systems that comprise entire computational networks. Yet the prize for making progress is to provide a clearer molecular understanding of the origins of human disease and to gain increased insight into the interactions of individuals with their environment (Weng et al., 1999).

In our laboratory, we are interested in the contributions to signaling networks made by inositol phosphates. One biological end-point that we focus on is the regulation of certain groups of Cl channels. This is of special interest to us because ion channels are the most rapid of all signaling entities, and so they provide particularly impressive examples of signal amplification. Minor fluctuations in the balance of opposing stimulatory and inhibitory signals can switch the conductance state of a single channel so as to influence the transmembrane movement of millions of ions per second (Clapham, 2001). The Cl channels that we study are activated by Ca2+ and by CaMKII, and they are inhibited by inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P4, Fig. 1). An important aspect to this regulatory process is the receptor-dependent regulation of intracellular levels of Ins(3,4,5,6)P4, in which a major participant is the physiologically reversible Ins(3,4,5,6)P4 1-kinase/Ins(1,3,4,5,6)P5 1-phosphatase (Fig. 2). It is this enzyme and the significance of its substrates that provides the major points of focus for this review.

Section snippets

Ins(3,4,5,6)P4 regulates the conductance of Ca2+-activated Cl channels in plasma membranes

It probably seems self-evident that we should first understand the significance of Ins(3,4,5,6)P4 as a cellular signal, so as to best appreciate the importance of the mechanisms that control the synthesis and metabolism of Ins(3,4,5,6)P4. It is somewhat ironic, therefore, that these milestones were transposed during the development of our insight into this signaling pathway. We actually had no idea what was the function of Ins(3,4,5,6)P4 when we (Menniti et al., 1990) first uncovered clues that

The receptor-regulated pathway of Ins(3,4,5,6)P4 synthesis

In the previous section we described the importance of Ins(3,4,5,6)P4 as a cellular signal. Thus, it should be clear that receptor-dependent control over steady-state levels of Ins(3,4,5,6)P4 plays an important cellular role. Nevertheless, the mass levels of Ins(3,4,5,6)P4 fluctuate within a fairly narrow range. There is approximately 1 μM Ins(3,4,5,6)P4 in “resting” cells and this rises to 4–10 μM after receptor-dependent activation of PLC (Ho and Shears, 2002). On the other hand, a very

Regulation of Ins(3,4,5,6)P4 levels by a reversible inositol phosphate kinase/phophatase

Our research into the control of Ins(3,4,5,6)P4 metabolism eventually came to center upon Ins(1,3,4)P3, a key downstream metabolite of Ins(1,4,5)P3 (Fig. 2). In a series of studies, we (Yang et al., 1999; Tan et al., 1997; Craxton et al., 1994) demonstrated that Ins(1,3,4)P3 competitively inhibits the activity of the Ins(3,4,5,6)P4 1-kinase. This changes the poise of the Ins(3,4,5,6)P4/Ins(1,3,4,5,6)P5 substrate cycle (Fig. 2). The Ins(1,3,4,5,6)P5 1-phosphatase activity is not inhibited by

The nature of the reversible Ins(3,4,5,6)P4/Ins(1,3,4)P3 kinase

The mammalian Ins(3,4,5,6)P4/Ins(1,3,4)P3 kinase is a 46 kDa protein; there appears to be only one isoform expressed in mammalian cells (Genbank NP_055031), although it is possible that different mRNA transcripts might be formed by alternate splicing, particularly in the 5′ non-translated region (Yang and Shears, 2000). The significance of there being multiple mRNAs is typically interpreted in terms of their differing in either stability or translatability, in order to modulate gene expression

Future directions

We still understand surprisingly little about the Ins(1,3,4)P3/Ins(3,4,5,6)P4 kinase. For example, the structure has not been solved. We have no idea which amino acid residues contribute to the active site of the protein, so we can only speculate as to the reaction mechanism that permits the kinase to be physiologically reversible. The proposed role of Ins(1,3,4,5,6)P5 as a phosphate donor to Ins(1,3,4)P3 (see Regulation of Ins(3,4,5,6)P4 Levels by a Reversible Inositol Phosphate

Summary

Inositol 3,4,5,6-tetrakisphate (Ins(3,4,5,6)P4) is an important cellular regulator of the conductance of certain types of chloride ion channels. A single enzyme is responsible for both synthesizing and metabolizing Ins(3,4,5,6)P4. This ezyme couples this bi-directional metabolism of Ins(3,4,5,6)P4 to the ongoing receptor-dependent hydrolysis of inositol lipids by phospholipase C. This review assesses the current state of our knowledge of this complex signal transduction process.

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