Atypical protein kinase C (aPKC) isozymes are unique in the protein kinase C (PKC) superfamily in that they are not regulated by the lipid second messenger diacylglycerol. Whether a different second messenger acutely controls their function is unknown. Here we show that the lipid mediator, sphingosine 1-phosphate (S1P), controls the cellular activity of aPKC. Using a genetically-encoded reporter we designed, aPKC-specific C Kinase Activity Reporter (aCKAR), we demonstrate that intracellular S1P activates aPKC. Biochemical studies reveal that S1P directly binds to the kinase domain of aPKC to relieve autoinhibitory constraints. In silico studies identify potential binding sites on the kinase domain, one of which was validated biochemically. Lastly, functional studies reveal that S1P-dependent activation of aPKC suppresses apoptosis in HeLa cells. Taken together, our data reveal a previously undescribed molecular mechanism for controlling the cellular activity of atypical PKC and identify a new molecular target for S1P.

Objective During rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) critically promote disease pathogenesis by aggressively invading the joint extracellular matrix. The focal adhesion kinase (FAK) signaling pathway is emerging as a contributor to RA FLS anomalous behavior. The receptor protein tyrosine phosphatase α (RPTPα), encoded by the PTPRA gene, is a key promoter of FAK signaling. Here we investigated whether RPTPα mediates FLS aggressiveness and RA pathogenesis. Methods Through RPTPα knockdown, we assessed FLS gene expression by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, invasion and migration in transwell assays, survival by Annexin V and propidium iodide staining, adhesion and spreading by immunofluorescence microscopy, and activation of signaling pathways by Western blotting of FLS lysates. Arthritis development was examined in Ptpra−/− mice using the K/BxN serum transfer model. The contribution of radiosensitive and radioresistant cells to disease was evaluated by reciprocal bone-marrow transplantation. Results RPTPα was enriched in the RA synovial lining. RPTPα knockdown impaired RA FLS survival, spreading, migration, invasiveness and responsiveness to platelet-derived growth factor, tumor necrosis factor and interleukin-1 stimulation. These phenotypes correlated with increased phosphorylation of SRC on inhibitory Y527 and decreased phosphorylation of FAK on stimulatory Y397. Treatment of RA FLS with an inhibitor of FAK phenocopied knockdown of RPTPα. Ptpra-deficient mice were protected from arthritis development, which was due to radioresistant cells. Conclusions By regulating phosphorylation of SRC and FAK, RPTPα mediates pro-inflammatory and pro-invasive signaling in RA FLS, correlating with promotion of disease in an FLS-dependent model of RA.

Spinocerebellar ataxia type 14 (SCA14) is a neurodegenerative disease caused by germline variants in the diacylglycerol (DAG)/Ca 2+ -regulated protein kinase Cγ (PKCγ), leading to Purkinje cell degeneration and progressive cerebellar dysfunction. Most of the identified mutations cluster in the DAG-sensing C1 domains. Here, we found with a FRET-based activity reporter that SCA14-associated PKCγ mutations, including a previously undescribed variant, D115Y, enhanced the basal activity of the kinase by compromising its autoinhibition. Unlike other mutations in PKC that impair its autoinhibition but lead to its degradation, the C1 domain mutations protected PKCγ from such down-regulation. This enhanced basal signaling rewired the brain phosphoproteome, as revealed by phosphoproteomic analysis of cerebella from mice expressing a human SCA14-associated H101Y mutant PKCγ transgene. Mutations that induced a high basal activity in vitro were associated with earlier average age of onset in patients. Furthermore, the extent of disrupted autoinhibition, but not agonist-stimulated activity, correlated with disease severity. Molecular modeling indicated that almost all SCA14 variants not within the C1 domain were located at interfaces with the C1B domain, suggesting that mutations in and proximal to the C1B domain are a susceptibility for SCA14 because they uniquely enhance PKCγ basal activity while protecting the enzyme from down-regulation. These results provide insight into how PKCγ activation is modulated and how deregulation of the cerebellar phosphoproteome by SCA14-associated mutations affects disease progression.

Protein kinase C (PKC) isozymes transduce myriad signals within the cell in response to the generation of second messengers from membrane phospholipids. The conventional isozyme PKCγ reversibly binds Ca2+ and diacylglycerol, which leads to an open, active conformation. PKCγ expression is typically restricted to neurons, but evidence for its expression in certain cancers has emerged. PKC isozymes have been labeled as oncogenes since the discovery that they bind tumor-promoting phorbol esters, however, studies of cancer-associated PKC mutations and clinical trial data showing that PKC inhibitors have worsened patient survival have reframed PKC as a tumor suppressor. Aberrant expression of PKCγ in certain cancers suggests a role outside the brain, although whether PKCγ also acts as a tumor suppressor remains to be established. On the other hand, PKCγ variants associated with spinocerebellar ataxia type 14 (SCA14), a neurodegenerative disorder characterized by Purkinje cell degeneration, enhance basal activity while preventing phorbol ester-mediated degradation. Although the basis for SCA14 Purkinje cell degeneration remains unknown, studies have revealed how altered PKCγ activity rewires cerebellar signaling to drive SCA14. Importantly, enhanced basal activity of SCA14-associated mutants inversely correlates with age of onset, supporting that enhanced PKCγ activity drives SCA14. Thus, PKCγ activity should likely be inhibited in SCA14, whereas restoring PKC activity should be the goal in cancer therapies. This review describes how PKCγ activity can be lost or gained in disease and the overarching need for a PKC structure as a powerful tool to predict the effect of PKCγ mutations in disease.

Spinocerebellar ataxia type 14 (SCA14) is a neurodegenerative disease caused by germline mutations in the diacylglycerol (DG)/Ca2+‐regulated protein kinase C gamma (PKCγ), leading to Purkinje cell degeneration and progressive cerebellar dysfunction. Curiously, the majority of the approximately 50 missense mutations identified in PKCγ cluster to the DG‐sensing C1A and C1B domains. Here, we use a genetically‐encoded FRET‐based C Kinase Activity Reporter (CKAR) to show that ataxia‐associated PKCγ mutants have higher basal activity in cells, and thus are less autoinhibited, than wild‐type enzyme. However, whereas reduced autoinhibition generally renders PKC sensitive to degradation, we show that mutations in the C1B domain allow translocation to membranes but protect PKCγ from phorbol ester‐induced down‐regulation. Indeed, deletion of the C1B domain prevents PKCγ down‐regulation with phorbol esters, potent ligands for the C1 domains. Strikingly, the degree of impaired autoinhibition correlates inversely with age of disease onset. Patients with the most severe mutation we examined (V138E) present with symptoms as young children, whereas symptoms in patients with the least severe mutation examined (D115Y) manifested in their 40s. To understand the structural basis of mutations outside the C1 domains, we generated a model of PKCγ using homology modeling and molecular docking. Mutations outside the C1 domains occur in regions also predicted to disrupt autoinhibition, including the pseudosubstrate, a predicted interface between the kinase and C1B domains, and the C‐terminal tail of PKCγ. Taken together, our data support a model in which SCA14 mutations enhance PKCγ activity without compromising stability. Furthermore, because many of the genetic causes of the 40+ types of SCA alter Ca2+ homeostasis, deregulated PKCγ activity may be a common cause for the disease. This raises the possibility that inhibition of PKCγ will be a potentially viable therapeutic target for SCA. Support or Funding Information NIH T32 GM007752