Project Summary
Extracellular-Signal Regulated Kinase (ERK), the terminal master kinase in the mitogen-activated
protein kinase (MAPK) signaling pathway, regulates a variety of critical cell processes, and its aberrant
activation contributes to the development of various cancers. In appendiceal, pancreatic, and colorectal
cancers, there is significant co-occurrence of activating mutations in the ERK pathway with activating mutations
in the cAMP/cAMP-dependent kinase (PKA) pathway, indicating cooperation between these two pathways in
cancer development. The exact mechanism of crosstalk between the two canonical signaling pathways has
been elusive. This proposal focuses on investigating the mechanisms by which PKA regulates ERK activity in a
context dependent manner.
Preliminary results in PC12 cells, a model cell line used for spatiotemporal ERK activity studies, indicate
both inhibitory and stimulatory regulation by PKA on plasma membrane localized ERK (pmERK) activity
depending on the initial activation of the two pathways. When PKA is activated before EGF activation of the
ERK pathway, the inhibitory effects of PKA on pmERK seem to dominate. On the other hand, when ERK
signaling is already active, PKA activation seems to sustain EGF stimulated pmERK activity. Previous studies
have shown PKA phosphorylation of the GTPase Rap1 causes two effects. One effect is that Rap1 leaves the
plasma membrane, which decreases pmERK activity. The other effect is that phosphorylated Rap1 interacts
with B-Raf, which sustains pmERK activity. Our preliminary results support the hypothesis that the GTPase
Rap1 mediates crosstalk between cAMP/PKA and pmERK activity.
To test this hypothesis, PKA and Rap1 biosensors will be co-imaged in single cells using an in-house
high-throughput, automated liquid handling microscope system. Various perturbations of the cAMP/PKA
pathway before and after EGF stimulation will be used to determine the temporal relationship between PKA
and Rap1. These experiments will then be repeated with Rap1 biosensors containing a mutated PKA
phosphorylation site to test whether removing PKA phosphorylation of Rap1 alone impedes the crosstalk. We
will develop a computational model to investigate whether the proposed mechanism is sufficient to replicate the
ERK activity observed in preliminary results and to predict how cancer-specific activating mutations in
upstream components of both pathways affect ERK activity. These model predictions will then be tested in
human colorectal cancer cell lines. The results of this work will uncover a previously unresolved mechanism of
PKA regulation of ERK activity, providing more information for the development of therapies for cancers driven
by aberrant ERK signaling.