Scaffolding, Multisite Phosphorylation and Other Aspects of Regulation in Signal Transduction

For cells to respond appropriately and timely to internal and external stimulus, they rely heavily on signaltransduction cascades to regulate gene and protein activity and response. Protein activity is oftenregulated by ligand binding, scaffolding, or by post-translational modifications such as multisitephosphorylation. The Mitogen-Activated Protein Kinases (MAPKs) are multisite proteins with critical rolesin development and various diseases. The c-Jun N-terminal Kinases (JNK) cascade is one of many MAPKcascades with multisite proteins. Here, we consider mathematical and experimental analyses tounderstand scaffolding and multisite modifications as protein regulation mechanisms.Scaffolding is a regulatory mechanism for signal transduction where scaffold proteins bind to manycomponents of a signaling pathway. MAPK scaffold proteins, such as IQ-motif-containing GTPaseactivatingprotein 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 manycomponents of a signaling pathway. Thus, it important to know which domains on IQGPA1 bind to whichMAP kinases for developing new therapies. Here, we show with in vitro binding assays that the IQ domainof IQGAP1 is both necessary and sufficient for binding to ERK1 and ERK2. Additionally, we show that theWW domain is neither necessary nor sufficient for binding to ERK1 or ERK2. These findings prompt a reevaluationof how IQGAP1 regulates MAPK cascade proteins.Protein phosphorylation also regulates a substrate’s enzymatic activity, location, stability, and/orinteractions with other proteins. Moreover, proteins that are regulated in this way often contain multiplemodification sites. In the JNK pathway, transcription factor c-Jun is a multisite substrate shown to regulatecell responses such as cell proliferation, apoptosis, and DNA repair. Understanding kinase-substratespecificity in MAPKs is crucial to advancing medical therapies for diseases. Phosphoproteomic studieshave 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 kinaseassays that the Docking site (D-site) in c-Jun plays a significant role in the phosphorylation of all native andnovel sites of c-Jun. Results indicate that the D-site is necessary for phosphorylation of native and novelsites 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 orbound by ligand have been observed to create an ultrasensitive dose response. Here, we consider thecontribution of the individual modification/binding sites to the activation process, and show how theirindividual 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, andassociate a so-called activation parameter to each site. Our analysis shows that ultrasensitivity generallydecreases with increasing activation parameter values and depends on their mean and not on theirvariability. Additionally, results suggest that a protein can increase its ultrasensitivity by evolving newsites. These results provide insights into the performance objectives of multiple modification/binding sitesand thus help gain a greater understanding of signaling and its role in diseases. Together, mathematicaland experimental analyses show promising insights into signal transduction regulation throughscaffolding, multisite PTMs, and docking