UC Irvine

For cells to respond appropriately and timely to internal and external stimulus, they rely heavily on signal

transduction cascades to regulate gene and protein activity and response. Protein activity is often

regulated by ligand binding, scaffolding, or by post-translational modifications such as multisite

phosphorylation. The Mitogen-Activated Protein Kinases (MAPKs) are multisite proteins with critical roles

in development and various diseases. The c-Jun N-terminal Kinases (JNK) cascade is one of many MAPK

cascades with multisite proteins. Here, we consider mathematical and experimental analyses to

understand scaffolding and multisite modifications as protein regulation mechanisms.

Scaffolding is a regulatory mechanism for signal transduction where scaffold proteins bind to many

components of a signaling pathway. MAPK scaffold proteins, such as IQ-motif-containing GTPaseactivating

protein 1 (IQGAP1), are promising targets for novel therapies against cancer and other diseases.

Scaffolding is a regulatory mechanism for signal transduction where scaffold proteins bind to many

components of a signaling pathway. Thus, it important to know which domains on IQGPA1 bind to which

MAP kinases for developing new therapies. Here, we show with in vitro binding assays that the IQ domain

of IQGAP1 is both necessary and sufficient for binding to ERK1 and ERK2. Additionally, we show that the

WW domain is neither necessary nor sufficient for binding to ERK1 or ERK2. These findings prompt a reevaluation

of how IQGAP1 regulates MAPK cascade proteins.

Protein phosphorylation also regulates a substrate’s enzymatic activity, location, stability, and/or

interactions with other proteins. Moreover, proteins that are regulated in this way often contain multiple

modification sites. In the JNK pathway, transcription factor c-Jun is a multisite substrate shown to regulate

cell responses such as cell proliferation, apoptosis, and DNA repair. Understanding kinase-substrate

specificity in MAPKs is crucial to advancing medical therapies for diseases. Phosphoproteomic studies

have provided insights into the conservation of phosphosites and their evolution across species. However,

not much is known about the constraints novel sites experience. Here, we demonstrate with in vitro kinase

assays that the Docking site (D-site) in c-Jun plays a significant role in the phosphorylation of all native and

novel sites of c-Jun. Results indicate that the D-site is necessary for phosphorylation of native and novel

sites of c-Jun by JNK2 enzyme.

Mathematical models are useful to study complex biological phenomena such as multisite proteins (i.e.,

proteins with n > 1 modifications sites). Proteins with multiple sites on which they can be modified or

bound by ligand have been observed to create an ultrasensitive dose response. Here, we consider the

contribution of the individual modification/binding sites to the activation process, and show how their

individual values affect the ultrasensitive behavior of the overall system. We use a generalized Monod-

Wyman-Changeux (MWC) model that allows for variable free energy contributions at distinct sites, and

associate a so-called activation parameter to each site. Our analysis shows that ultrasensitivity generally

decreases with increasing activation parameter values and depends on their mean and not on their

variability. Additionally, results suggest that a protein can increase its ultrasensitivity by evolving new

sites. These results provide insights into the performance objectives of multiple modification/binding sites

and thus help gain a greater understanding of signaling and its role in diseases. Together, mathematical

and experimental analyses show promising insights into signal transduction regulation through

scaffolding, multisite PTMs, and docking.