By Dean A. Haycock

Special To BioWorld Today

If only it were simpler. If only there were fewer chemical players in the pathways that regulate cell division. If only . . .

Fortunately, wishful thinkers usually don't become successful researchers. Accepting the complexity of biochemical puzzles like those that control the growth of both healthy and malignant cells is a job requirement.

A good example of the challenges researchers face is the regulation of an enzyme called protein kinase B (PKB). PKB, which is over-expressed in many human tumors, is a proto-oncogene. Proto-oncogenes are proteins which, in some of their forms, are linked to tumor formation.

PKB influences cell division after getting a signal from growth factor receptors on the cell membrane. It also may be involved in programmed cell death.

"For any tumor to become well established you need to induce some way of inhibiting cell death which is induced by an activated oncogene," said David Stokoe, assistant research biochemist at the University of California at San Francisco Cancer Research Institute.

Influencing cell proliferation and death is a big responsibility, of course. Mess this job up and cell division can run uncontrolled. That means cancer. So PKB is itself subject to elaborate controls; it is regulated indirectly by another enzyme called PI3K. Scientists already knew PI3K played an important role in regulating cell division. Its product is a lipid, PtdIns(3,4,5)P₃, which acts as a "second messenger" carrying messages from a growth factor, the "first messenger." It may also have roles in cancer and, perhaps, diabetes.

Researchers recently have come to suspect that this second messenger plays dual roles in the regulation of cell growth. One is to promote the attachment of proteins to membranes and the other is to activate protein kinases. Kinases are enzymes like PKB that affect other proteins by attaching phosphate groups to them. Still, the details of how PtdIns(3,4,5)P3 regulated cell growth under control were unknown.

They are better understood now, in part due to a paper in the July 25, 1997, issue of Science, "Dual role of phosphatidylinositol-3,4,5-triphosphate in the activation of protein kinase B." First author David Stokoe was a postdoctoral fellow at Onyx Pharmaceuticals Inc., in Richmond, Calif., when he and his co-authors showed how PtdIns(3,4,5)P₃ regulates PKB.

The complicated control system Stokoe and his colleagues describe includes two different but cooperative methods for regulating the oncogene PKB. First, the lipid second messenger PtdIns(3,4,5)P₃ may ensure that PKB is correctly located in the cell membrane, where it does its job of phosphorylating other proteins. Second, the lipid, by binding to PKB, alters the shape of the PKB molecule.

"It binds to PKB and changes its conformation such as to allow it to become a better substrate for USK," Stokoe said.

USK, or upstream kinase, is a new enzyme Stokoe and his co-workers identified and described in the paper. Upstream refers to the flow of information from growth factor to receptor to second messengers to molecules that initiate an action such as cell division. The researchers also found that PtdIns(3,4,5)P₃ directly activates USK.

Why two regulatory mechanism when one might do?

"It is not the first pathway [to be described] where one molecule is exerting its effects at multiple levels," Stokoe said.

He cites the example of an enzyme called AMP-activated protein kinase, for which there are at least four different methods for regulating its activity.

"It just indicates how complex these pathways can be," Stokoe said.

USK appears to be similar to an enzyme called PDK1, which was described recently in Current Biology.

"PDK1 and USK look to be basically in the same family," Stokoe said.

The findings highlight the variety of the regulatory mechanisms cells use to control the activation of PKB. The PKB regulatory pathway draws on most of the known methods used by cells to control its messengers. These include protein phosphorylation of two varieties (tyrosine and serine/threonine), phosphorylation of second messenger lipids, protein recruitment and allosteric activation.

The finer description of the PKB oncogene's regulation moves biomedical researchers a bit closer to understanding, and perhaps better manipulating, processes underlying cell regulation and cancer.

Because subsets of tumors over-express PKB, inhibitors of its regulatory pathways have become inviting targets for the biotech industry.

"Even though only, say, 10 percent of tumors examined so far actually amplify PKB at the gene level, there could be a lot higher percentage of tumors which have different defects in the pathway," Stokoe said.

Stokoe suggests that point mutants in USK, defects in the downstream pathway or other factors that increase activity in this pathway could prevent tumor cells from undergoing apoptosis.

"The more components in a pathway you can identify, the greater the chance of getting a drug that will selectively inhibit one component. The identification of an upstream kinase will now give at least one more thing to screen for," Stokoe said. *