Your morning Danish sets off a train of cellular eventsthat convert it to sugar, then releases it as energy or storesit as fat. That influx of glucose into your blood acts as awake-up call for the insulin genes in the beta cells of thepancreatic Islets of Langerhans.

So far, that's high school biology. Trying to understand atthe nitty grittiest molecular level how that signal entersthe cell, makes its way through the cytoplasm to thenucleus, and actually triggers the gene to transcribe DNAto RNA to insulin, is keeping a lot of cell biologists andbiochemists busy.

One who wears both hats is Andrew Larner, a seniorscientist at the FDA's Center for Biologics Evaluationand Research. He invokes that insulin protein pathwaymerely as an example of how intracellular signalingpathways work in general _ as far as he and others nowknow.

"The key event by which insulin regulates glucosemetabolism," Larner told BioWorld Today, "is byphosphorylation or dephosphorylation of the glycogen-synthesizing enzyme. Putting on the phosphate groupsinitiates its breakdown into sugar, which then releases itfrom the cells in the body as energy."

Phosphorylation itself is a key event in every cell, whereit drives the relay race of enzymes along the cellularsignaling pathways.

Larner's paper in the current issue of Science, dated Sept.22, 1995, reports one of the latest advances in penetratingthe still mysterious maze of cell signaling. Its title:"Requirement for MAP Kinase [ERK2] activity ininterferon-a- and interferon-b-stimulated gene expressionthrough STAT proteins."

"The underlying hypothesis of this work," Larner said, "isthat you've got a set of genes that make proteins, whichare rapidly activated by the action of cytokines in the cell.Production of the proteins initiates a cascade of signalingevents that leads to the genes' ultimate biologicalresponse."

Interferon Models `Go' On Cell-Signaling Pathway

In their latest experiment, reported in Science, Larner andhis co-authors took interferon-alpha as their modelcytokine, (with its receptor donated by Chris Benjamin,of Biogen Inc. in Cambridge, Mass.) "As is the case ofmany other cytokines," Larner observed, "interferon[IFN] affects cell growth and differentiation.

"So the idea is," he continued, "if one can understandhow these proteins are expressed rapidly as an earlysignaling event, one is going to have a handle on what'sneeded to get the signaling going. That is, getting thecytokine's message from outside the cell into itscytoplasm and nucleus."

Larner's team started with a cultured human myelomacell line and primary human fibroblasts. "We triggeredthem with IFN," he recounted, "then used antibodies toanalyze the rapid events that happen after that treatment,the activation of kinases, such as MAP kinase, geneexpression, and the association of such kinases withtranscription of DNA to RNA."

Kinases are enzymes that phosphorylate a substrate, suchas another enzyme. MAP _ mitogen-activated proteinkinase _ "appears to be a switch kinase," Larnerobserved, "essential for regulating growth anddevelopment in a number of cellular signaling cascades. Itswitches from using tyrosine phosphorylation, whichactivates MAP itself, to phosphorylation of anotheramino acid, serine.

"As we described in the Science paper," Larner pointedout, "serine as well as tyrosine must have phosphategroups stuck on, to allow the cytokine-stimulated genesto be activated, and initiate RNA transcription."

This demonstration of a two-way traffic intersectionalong the cell's signaling pathway, Larner went on,"parallels or complements or adds to an earlier paper byJames Darnell of Rockefeller University, which came outa couple of months ago. He showed that you need serinephosphorylation, and speculated that it would involveMAP kinase.

"We showed," Larner continued, "that you do indeedneed MAP, and if you knock it out in the cell, you knockout the ability to transcribe these genes."

From Molecular Level To Bedside _ Some Day

The next piece in the signaling pathway puzzle thatLarner and his team are trying to fit in is whether or notMAP kinase activation's pathway is dependent on the Rafpathway. Raf, which, like Ras, when overexpressed isoncogenic, initiates a pathway cascade that activatesMAP.

"If it turns out to be independent," Larner observed, "itsays that there's another set of kinase-activatingmechanisms that do not require Raf to initiate MAP.

He suggested that there may be therapeutic payoffs in thispursuit, "but a ways off. It implies that what wasoriginally thought to be two distinct signaling pathways,regulated by a common set of growth factors _ in thiscase, IFN _ are not distinct. They talk to each other; theymodulate each other's activities."

He concluded: "Understanding how that occurs at thebiochemical and molecular levels is where things areheaded therapeutically." n

-- David N. Leff Science Editor

(c) 1997 American Health Consultants. All rights reserved.