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Open Access Publications from the University of California

Scholarly Works (37 results)

  • Article
  • Peer Reviewed
UC Irvine Previously Published Works (2015)
MAPKs bind to many of their upstream regulators and downstream substrates via a short docking motif (the D-site) on their binding partner. MAPKs that are in different families (e.g. ERK, JNK, and p38) can bind selectively to D-sites in their authentic substrates and regulators while discriminating against D-sites in other pathways. Here we demonstrate that the short hydrophobic region at the distal end of the D-site plays a critical role in determining the high selectivity of JNK MAPKs for docking sites in their cognate MAPK kinases. Changing just 1 or 2 key hydrophobic residues in this submotif is sufficient to turn a weak JNK-binding D-site into a strong one, or vice versa. These specificity-determining differences are also found in the D-sites of the ETS family transcription factors Elk-1 and Net. Moreover, swapping two hydrophobic residues between these D-sites switches the relative efficiency of Elk-1 and Net as substrates for ERK versus JNK, as predicted. These results provide new insights into docking specificity and suggest that this specificity can evolve rapidly by changes to just 1 or 2 amino acids.
    • Article
    • Peer Reviewed
    UC Irvine Previously Published Works (2004)

    Anthrax lethal toxin is the major cause of death in systemic anthrax. Lethal toxin consists of two proteins: protective antigen and LF (lethal factor). Protective antigen binds to a cell-surface receptor and transports LF into the cytosol. LF is a metalloprotease that targets MKKs [MAPK (mitogen-activated protein kinase) kinases]/MEKs [MAPK/ERK (extracellular-signal-regulated kinase) kinases], cleaving them to remove a small N-terminal stretch but leaving the bulk of the protein, including the protein kinase domain, intact. LF-mediated cleavage of MEK1 and MKK6 has been shown to inhibit signalling through their cognate MAPK pathways. However, the precise mechanism by which this proteolytic cleavage inhibits signal transmission has been unclear. Here we show that the C-terminal LF-cleavage products of MEK1, MEK2, MKK3, MKK4, MKK6 and MKK7 are impaired in their ability to bind to their MAPK substrates, suggesting a common mechanism for the LF-induced inhibition of signalling

      Cover page: Anthrax lethal factor-cleavage products of MAPK (mitogen-activated protein kinase) kinases exhibit reduced binding to their cognate MAPKs
      • Article
      • Peer Reviewed
      UC Irvine Previously Published Works (2017)
      Mitogen-activated protein kinase (MAPK) scaffold proteins, such as IQ motif containing GTPase activating protein 1 (IQGAP1), are promising targets for novel therapies against cancer and other diseases. Such approaches require accurate information about which domains on the scaffold protein bind to the kinases in the MAPK cascade. Results from previous studies have suggested that the WW domain of IQGAP1 binds to the cancer-associated MAPKs ERK1 and ERK2, and that this domain might thus offer a new tool to selectively inhibit MAPK activation in cancer cells. The goal of this work was therefore to critically evaluate which IQGAP1 domains bind to ERK1/2. Here, using quantitative in vitro binding assays, we show that the IQ domain of IQGAP1 is both necessary and sufficient for binding to ERK1 and ERK2, as well as to the MAPK kinases MEK1 and MEK2. Furthermore, we show that the WW domain is not required for ERK-IQGAP1 binding, and contributes little or no binding energy to this interaction, challenging previous models of how WW-based peptides might inhibit tumorigenesis. Finally, we show that the ERK2-IQGAP1 interaction does not require ERK2 phosphorylation or catalytic activity and does not involve known docking recruitment sites on ERK2, and we obtain an estimate of the dissociation constant (Kd ) for this interaction of 8 μm These results prompt a re-evaluation of published findings and a refined model of IQGAP scaffolding.
        Cover page: The WW domain of the scaffolding protein IQGAP1 is neither necessary nor sufficient for binding to the MAPKs ERK1 and ERK2
        • Article
        • Peer Reviewed
        UC Irvine Previously Published Works (2022)
        Crosstalk between the Hedgehog and MAPK signaling pathways occurs in several types of cancer and contributes to clinical resistance to Hedgehog pathway inhibitors. Here we show that MAP kinase-mediated phosphorylation weakens the binding of the GLI1 transcription factor to its negative regulator SUFU. ERK2 phosphorylates GLI1 on three evolutionarily conserved target sites (S102, S116, and S130) located near the high-affinity binding site for SUFU; these phosphorylations cooperate to weaken the affinity of GLI1-SUFU binding by over 25-fold. Phosphorylation of any one, or even any two, of the three sites does not result in the level of SUFU release seen when all three sites are phosphorylated. Tumor-derived mutations in R100 and S105, residues bordering S102, also diminish SUFU binding, collectively defining a novel evolutionarily conserved SUFU affinity-modulating region. In cultured mammalian cells, GLI1 variants containing phosphomimetic substitutions of S102, S116, and S130 displayed an increased ability to drive transcription. We conclude that multisite phosphorylation of GLI1 by ERK2 or other MAP kinases weakens GLI1-SUFU binding, thereby facilitating GLI1 activation and contributing to both physiological and pathological crosstalk.
          Cover page: ERK2 MAP kinase regulates SUFU binding by multisite phosphorylation of GLI1
          • Article
          • Peer Reviewed
          UC Irvine Previously Published Works (2023)
          IQGAP1 is a multi-domain cancer-associated protein that serves as a scaffold protein for multiple signaling pathways. Numerous binding partners have been found for the calponin homology, IQ and GAP-related domains in IQGAP1. Identification of a binding partner for its WW domain has proven elusive, however, even though a cell-penetrating peptide derived from this domain has marked anti-tumor activity. Here, using in vitro binding assays with human proteins and co-precipitation from human cells, we show that the WW domain of human IQGAP1 binds directly to the p110α catalytic subunit of phosphoinositide 3-kinase (PI3K). In contrast, the WW domain does not bind to ERK1/2, MEK1/2, or the p85α regulatory subunit of PI3K when p85α is expressed alone. However, the WW domain is able to bind to the p110α/p85α heterodimer when both subunits are co-expressed, as well as to the mutationally activated p110α/p65α heterodimer. We present a model of the structure of the IQGAP1 WW domain, and experimentally identify key residues in the hydrophobic core and beta strands of the WW domain that are required for binding to p110α. These findings contribute to a more precise understanding of IQGAP1-mediated scaffolding, and of how IQGAP1-derived therapeutic peptides might inhibit tumorigenesis.
            Cover page: The WW domain of IQGAP1 binds directly to the p110α catalytic subunit of PI 3-kinase.
            • Article
            • Peer Reviewed
            UC Irvine Previously Published Works (2010)
            Abstract Background Interconnected cell signaling pathways are able to efficiently and accurately transmit a multitude of different signals, despite an inherent potential for undesirable levels of cross-talk. To ensure that an appropriate response is produced, biological systems have evolved network-level mechanisms that insulate pathways from crosstalk and prevent 'leaking' or 'spillover' between pathways. Many signaling pathways have been shown to respond in an ultrasensitive (switch-like) fashion to graded input, and this behavior may influence specificity. The relationship of ultrasensitivity to signaling specificity has not been extensively explored. Results We studied the behavior of simple mathematical models of signaling networks composed of two interconnected pathways that share an intermediate component, asking if the two pathways in the network could exhibit both output specificity (preferentially activate their own output) and input fidelity (preferentially respond to their own input). Previous results with weakly-activated pathways indicated that neither mutual specificity nor mutual fidelity were obtainable in the absence of an insulating mechanism, such as cross-pathway inhibition, combinatorial signaling or scaffolding/compartmentalization. Here we found that mutual specificity is obtainable for hyperbolic or ultrasensitive pathways, even in the absence of an insulating mechanism. However, mutual fidelity is impossible at steady-state, even if pathways are hyperbolic or ultrasensitive. Nevertheless, ultrasensitivity does provide advantages in attaining specificity and fidelity to networks that contain an insulating mechanism. For networks featuring cross-pathway inhibition or combinatorial signaling, ultrasensitive activation can increase specificity in a limited way, and can only be utilized by one of the two pathways. In contrast, for networks featuring scaffolding/compartmentalization, ultrasensitive activation of both pathways can dramatically improve network specificity. Conclusions There are constraints to obtaining performance objectives associated with signaling specificity; such constraints may have influenced the evolution of signal transduction networks. Notably, input fidelity (preferential response to an authentic input) is a more difficult objective to achieve than output specificity (preferential targeting to an authentic output). Indeed, mutual fidelity is impossible in the absence of an insulating mechanism, even if pathways are ultrasensitive. Ultrasensitivity does, however, significantly enhance the performance of several insulating mechanisms. In particular, the ultrasensitive activation of both pathways can provide substantial improvement to networks containing scaffolding/compartmentalization.
              Cover page: Ultrasensitive responses and specificity in cell signaling
              • Article
              • Peer Reviewed
              UC Irvine Previously Published Works (2010)
              Background: Interconnected cell signaling pathways are able to efficiently and accurately transmit a multitude of different signals, despite an inherent potential for undesirable levels of cross-talk. To ensure that an appropriate response is produced, biological systems have evolved network-level mechanisms that insulate pathways from crosstalk and prevent 'leaking' or 'spillover' between pathways. Many signaling pathways have been shown to respond in an ultrasensitive (switch-like) fashion to graded input, and this behavior may influence specificity. The relationship of ultrasensitivity to signaling specificity has not been extensively explored. Results: We studied the behavior of simple mathematical models of signaling networks composed of two interconnected pathways that share an intermediate component, asking if the two pathways in the network could exhibit both output specificity (preferentially activate their own output) and input fidelity (preferentially respond to their own input). Previous results with weakly-activated pathways indicated that neither mutual specificity nor mutual fidelity were obtainable in the absence of an insulating mechanism, such as cross-pathway inhibition, combinatorial signaling or scaffolding/compartmentalization. Here we found that mutual specificity is obtainable for hyperbolic or ultrasensitive pathways, even in the absence of an insulating mechanism. However, mutual fidelity is impossible at steady-state, even if pathways are hyperbolic or ultrasensitive. Nevertheless, ultrasensitivity does provide advantages in attaining specificity and fidelity to networks that contain an insulating mechanism. For networks featuring cross-pathway inhibition or combinatorial signaling, ultrasensitive activation can increase specificity in a limited way, and can only be utilized by one of the two pathways. In contrast, for networks featuring scaffolding/compartmentalization, ultrasensitive activation of both pathways can dramatically improve network specificity. Conclusions: There are constraints to obtaining performance objectives associated with signaling specificity; such constraints may have influenced the evolution of signal transduction networks. Notably, input fidelity (preferential response to an authentic input) is a more difficult objective to achieve than output specificity (preferential targeting to an authentic output). Indeed, mutual fidelity is impossible in the absence of an insulating mechanism, even if pathways are ultrasensitive. Ultrasensitivity does, however, significantly enhance the performance of several insulating mechanisms. In particular, the ultrasensitive activation of both pathways can provide substantial improvement to networks containing scaffolding/compartmentalization.
              • 1 supplemental PDF
              Cover page: Ultrasensitive Responses and Specificity in Cell SignalingCreative Commons 'BY' version 4.0 license
              • Thesis
              • Peer Reviewed
              UC Irvine Electronic Theses and Dissertations (2020)

              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.

                Cover page: Scaffolding, Multisite Phosphorylation and Other Aspects of Regulation in Signal Transduction
                • Article
                • Peer Reviewed
                UC Irvine Previously Published Works (2011)
                • 1 supplemental PDF
                • 1 supplemental video
                Cover page: Noise filtering tradeoffs in spatial gradient sensing and cell polarization responseCreative Commons 'BY' version 4.0 license
                • Article
                • Peer Reviewed
                UC San Diego Previously Published Works (2005)

                Background: Associations between physical and emotional stress and negative mood states have been documented in a variety of populations. In military personnel, more physical symptoms and decrements in ability, to perform critical tasks have been shown to accompany such stress-induced negative mood. Most research in this area has focused on immediate effects of stress on mood. Purpose: We wondered what immediate mood effects strenuous training would have on Marines, what mood effects would endure 30 and 90 days after completion of training, and how mood scores would compare with normative data. Methods: Sixty male Marines (M age = 19 years, range = 18-28) completed the Profile of Mood States (POMS) at multiple time points before and after participating in a 30-day, cold weather, high-altitude field training exercise. Anthropometric measurements were taken at the same time points. Results: The Marines reported significant increases in POMS scores from baseline to completion of training, most of which endured up to 90 days. The anger and fatigue scores reported by the Marines were comparable to adult male psychiatric outpatient norms. Conclusions: Rigorous training in challenging environments may result in enduring negative moods that approach levels of clinical significance and may have implications for readiness for duty and performance of critical tasks. Behavioral medicine interventions may be helpful in military populations to reduce the impact of negative mood.

                  Cover page: Negative mood endures after completion of high-altitude military training
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