Protein kinase C (PKC) enzymes are a family of kinases that mediate signal transduction originating at the cell surface. Most cell membranes can contain functional PKC enzymes. Aberrations in the PKC life cycle may result in cellular damage and dysfunction. For example, some cancerous cells exhibit alterations in PKC activity. Here, we use a systems biology approach to describe a molecular model of the PKC life cycle. Understanding the PKC life cycle is necessary to identify new drug targets. The PKC life cycle is composed of three key regulatory processes: maturation, activation, and termination. These processes precisely control PKC enzyme levels. This model describes the fate of PKC during de novo synthesis and PKC’s lipid-mediated activation cycle. We utilize a systems biology approach to show the PKC life cycle is controlled by multiple phosphorylation and dephosphorylation events. PKC processing events can be divided into two types: maturation via processing of newly synthesized enzyme and secondary messenger-dependent activation of dormant, but catalytically competent enzyme. Newly synthesized PKC enzyme is constitutively processed through three ordered phosphorylations and stored in the cytosol as a stable, signaling-competent inactive and autoinhibited molecule. Upon extracellular stimulation, diacylglycerol (DAG) and calcium ion (Ca²⁺) generated at the membrane bind PKC. PKC then undergoes cytosol-to-membrane translocation and subsequent activation. Our model shows that, once activated, PKC is prone to dephosphorylation and subsequent degradation. This model also describes the role of HSP70 in stabilization and re-phosphorylation of dephosphorylated PKC, replenishing the PKC pool. Our model shows how the PKC pool responds to different intensities of extracellular stimuli? We show that blocking PHLPP dephosphorylation replenishes the PKC pool in a dose-dependent manner. This model provides a comprehensive understanding of PKC life cycle regulation.

Rationale: Heart failure (HF) is marked by dampened cardiac contractility. A mild therapeutic target that improves contractile function without desensitizing the β-adrenergic system during HF may improve cardiac contractility and potentially survival. Inhibiting protein kinase C α (PKCα) activity may fit the criteria of a therapeutic target with milder systemic effects that still boosts contractility in HF patients. PKCα activity has been observed to increase during HF. This increase in PKCα activity is perplexing because it is also accompanied by up-regulation of a molecular braking mechanism. Objective: I aim to explore how PKCα activity can be increased and maintained during HF despite the presence of a molecular braking mechanism. Methods and Results: Using a computational approach, I show that the local diacylglycerol (DAG) signaling is regulated through a two-compartment signaling system in cardiomyocytes. These results imply that after massive myocardial infarction (MI), local homeostasis of DAG signaling is disrupted. The loss of this balance leads to prolonged activation of PKCα, a key molecular target linked to LV remodeling and dysfunctional filling and ejection in the mammalian heart. This study also proposes an explanation for how DAG homeostasis is regulated during normal systolic and diastolic cardiac function. Conclusions: I developed a novel two-compartment computational model for regulating DAG homeostasis during Ang II-induced heart failure. This model provides a promising tool with which to study mechanisms of DAG signaling regulation during heart failure. The model can also aid in identification of novel therapeutic targets with the aim of improving the quality of life for heart failure patients.

Canonical transient receptor potential channels (TRPC3) may play a pivotal role in the development and viability of dendritic arbor in Purkinje neurons. This is a novel postsynaptic channel for glutamatergic synaptic transmission. In the cerebellum, TRPC3 appears to regulate functions relating to motor coordination in a highly specific manner. Gain of TRPC3 function is linked to significant alterations in the density and connectivity of dendritic arbor in Purkinje neurons. TRPC3 signals downstream of class I metabotropic glutamate receptors (mGluR1). Moreover, diacylglycerol (DAG) can directly bind and activate TRPC3 molecules. Here, we investigate a key question: How can the activity of the TRPC3 channel be regulated in Purkinje neurons? We also explore how mGluR1 activation, Ca²⁺influx, and DAG homeostasis in Purkinje neurons can be linked to TRPC3 activity modulation. Through systems biology approach, we show that TRPC3 activity can be modulated by a Purkinje cell (PC)–specific local signalosome. The assembly of this signalosome is coordinated by DAG generation after mGluR1 activation. Our results also suggest that purinergic receptor activation leads to the spatial and temporal organization of the TRPC3 signaling module and integration of its key effector molecules such as DAG, PKCγ, DGKγ, and Ca²⁺into an organized local signalosome. This signaling machine can regulate the TRPC3 cycling between active, inactive, and desensitized states. Precise activity of the TRPC3 channel is essential for tightly regulating the Ca²⁺entry into PCs and thus the balance of lipid and Ca²⁺signaling in Purkinje neurons and hence their viability. Cell-type–specific understanding of mechanisms regulating TRPC3 channel activity could be key in identifying therapeutic targeting opportunities.

Spinocerebellar ataxia type 14 (SCA14) is an autosomal neurodegenerative disease clinically characterized by progressive ataxia in the patient’s gait, accompanied by slurred speech and abnormal eye movements. These symptoms are linked to the loss of Purkinje cells (PCs), which leads to cerebellar neurodegeneration. PC observations link the mutations in PRKCG gene encoding protein kinase C γ (PKCγ) to SCA14. Observations also show that the link between PKCγ and SCA14 relies on a gain-of-function mechanism, and, in fact, both positive and negative regulation of PKCγ expression and activity may result in changes in cellular number, size, and complexity of the dendritic arbors in PCs. Here, through a systems biology approach, we investigate a key question relating to this system: why is PKCγ membrane residence time reduced in SCA14 mutant PCs compared to wild-type (WT) PCs? In this study, we investigate this question through two contrasting PKCγ signaling models in PCs. The first model proposed in this study describes the mechanism through which PKCγ signaling activity may be regulated in WT PCs. In contrast, the second model explores how mutations in PKCγ signaling affect the state of SCA14 in PCs. Numerical simulations of both models show that, in response to extracellular stimuli-induced depolarization of the membrane compartment, PKCγ and diacylglycerol kinase γ (DGKγ) translocate to the membrane. Results from our computational approach indicate that, for the same set of parameters, PKCγ membrane residence time is shorter in the SCA14 mutant model compared to the WT model. These results show how PKCγ membrane residence time is regulated by diacylglycerol (DAG), causing translocated PKCγ to return to the cytosol as DAG levels drop. This study shows that, when the strength of the extracellular signal is held constant, the membrane lifetime of mutant PKCγ is reduced. This reduction is due to the presence of constitutively active mutant PKCγ in the cytosol. Cytosolic PKCγ, in turn, leads to phosphorylation and activation of DGKγ while it is still residing in the cytosol. This effect occurs even during the resting conditions. Thus, the SCA14 mutant model explains that, when both DAG effector molecules are active in the cytosol, their interactions in the membrane compartment are reduced, critically influencing PKCγ membrane residence time.

Protein kinase C (PKC) enzymes are a family of kinases that mediate signal transduction originating at the cell surface. Most cell membranes can contain functional PKC enzymes. Aberrations in the PKC life cycle may result cellular damage and dysfunction. For example, some cancerous cells exhibit alterations in PKC activity. Here, we use a systems biology approach to describe a molecular model of the PKC life cycle. Understanding the PKC life cycle is necessary to identify new anticancer drug targets. The PKC life cycle is composed of three key regulatory processes: maturation, activation and termination. These processes precisely control PKC enzyme levels. This model describes the fate of PKC during de novo synthesis and PKC’s lipid-mediated activation cycle. We utilize a systems biology approach to show the PKC life cycle is controlled by multiple phosphorylation and dephosphorylation events. PKC processing events can be divided into two types: maturation via processing of newly synthesized enzyme and secondary messenger-dependent activation of dormant, but catalytically-competent enzyme. Newly synthesized PKC enzyme is constitutively processed through three ordered phosphorylations and stored in the cytosol as a stable, signaling-competent inactive and autoinhibited molecule. Upon extracellular stimulation, diacylglycerol (DAG) and calcium ion (Ca ⁺² ) generated at the membrane bind PKC. PKC then undergoes cytosol-to-membrane translocation and subsequent activation. Our model shows that, once activated, PKC is prone to dephosphorylation and subsequent degradation. This model also describes the role of HSP70 in stabilization and re-phosphorylation of dephosphorylated PKC, replenishing the PKC pool. Our model shows how the PKC pool responds to different intensities of extracellular stimuli? We show that blocking PHLPP dephosphorylation replenishes the PKC pool in a dose-dependent manner. This model provides a comprehensive understanding of PKC life cycle regulation.

How can synaptic efficacies be maintained despite the fast turnover of proteins at synapses? Partially, we know that the synthesis of new proteins is essential for the induction of the late, long-lasting phase of long-term potentiation (L-LTP). Recent experiments suggest that the concentration of protein kinase Mζ (PKMζ) is increased during L-LTP and that inhibiting the PKMζ activity during the maintenance phase can effectively reverse L-LTP. Experiments have also shown that phosphorylation is necessary for the activation of PKMζ. However, it is not clear what mechanism maintains the level and activity of PKMζ despite protein turnover and phosphatase activity. Using a mathematical modeling framework, I examine the hypothesis that the activity of PKMζ is sustained through a local switching mechanism. The model for the switching mechanism is motivated by several experimental observations: 1) PKMζ has two phosphorylation sites; one is mediated by another constitutively active kinase, Phosphoinositide-dependent kinase 1 PDK1 (T410) and is essential for its activity, and another is an autophosphorylation site, T560. 2) The phosphorylation of PKMζ increases its stability and the doubly phosphorylated PKMζ has a significantly longer lifetime than the unphosphorylated and singly phosphorylated states of PKMζ. 3) The doubly phosphorylated PKMζ also regulates the new synthesis of PKMζ through a translation feedback loop. The present study implemented a mass action model consistent with these observations. The results show that such a model can be bistable and that L-LTP induction produces an increase in the total amount of PKMζ at active synapses. The increase in PKMζ concentration was maintained through the regulation of new protein synthesis by PKMζ. The results also show that blocking the activity of PKMζ in a dose-dependent manner can effectively abolish the increase in the total amount of PKMζ, which is consistent with the effect that the PKMζ inhibitor zeta inhibitory peptide (ZIP) has experimentally demonstrated. The model is consistent with available experimental results regarding the phosphorylation levels of PKMζ and the temporal aspects of blocking experiments and produces a new prediction.