Similar exclusions of the GTPase Ras and PLC were made on the basis of the failure of inhibitors of these enzymes to affect PACAP-induced PC12 cell neuritogenesis ( 1, 7, 8). Protein kinase C (PKC) can be excluded because inhibitors of PKC do not block PACAP-induced neuritogenesis ( 7), and this reduces the number of potential pathways to 108. Akt can be excluded, because inhibition of phosphatidylinositol 3-kinase (PI3K), which activates Akt, does not abolish PACAP-induced neuritogenesis in PC12 cells ( 6) this reduces the number of potential pathways from 165 to 141. In this example, the goal is to identify the possible pathways from PACAP to neuritogenesis in PC12 cells and to winnow the list by excluding components on the basis of available experimental data. The power of pathFinder is that it provides a “working memory” that can be used to pare down the list of potential pathways to a list of likely pathways for a given cell type or process.
Slide 9: The effect of excluding elements on pathway output
PATH FINDER TOOL SERIES
Likewise, activation of extracellular signal–regulated kinase (ERK) by the guanosine triphosphatase (GTPase) Rap1 through the cyclic AMP–regulated guanine nucleotide exchange factor cAMP-GEFII (also known as Epac) is incorporated as a series of dyads (lines 5 through 8 of output), even though in PC12 cells Rap1 activation by cAMPGEFII does not lead to ERK activation ( 1). There may be cells in which PACAP causes Ca 2+mob without the G q-mediated activation of phospholipase C–β (PLC-β) ( 5). For example, PACAP is thought to cause calcium mobilization (Ca 2+mob) through activation of G q downstream of the PACAP receptor (PACAP-R) ( 3) but many reports document downstream effects of PACAP-induced Ca 2+mob, without mentioning either the specific PACAP receptor isoform or G protein involved in signaling ( 4) (compare lines 2 and 3 of output). Some redundancy is built into the analysis because of differing levels of specificity in the literature. This query reveals 165 potential pathways, of which only the first 23 are shown. PACAP activates the G protein–coupled receptor (GPCR) PAC1 on PC12 cells, which leads to neurite formation, a process called neuritogenesis. The neuropeptide PACAP was selected as the activated element, and neurites were selected as the target element. The value of this static network analysis tool is illustrated by the predication from pathFinder analysis of a novel cyclic AMP–dependent, protein kinase A–independent signaling pathway in neuroendocrine cells, which has been experimentally confirmed.
PATH FINDER TOOL ACTIVATOR
Missing elements in a pathway connecting the activator or initiator and target can also be inferred from a null pathway result. One or more intermediate pathway components can be excluded to simulate the use of pharmacological inhibitors or genetic deletion (knockout).
pathFinder can identify qualitatively all possible signal transduction pathways connecting any starting component and target within a database of two-component pathways (directional dyads). Among these, pathFinder is a unique tool that can find signal transduction pathways between first, second, or nth messengers and their targets within the cell.
Many online tools are used to cluster genes in various ways or to assign gene products to signal transduction pathways.
The study of signal transduction is becoming a de facto part of the analysis of gene expression and protein profiling techniques.
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