PHLPP Signaling in Immune Cells.

Pleckstrin homology domain leucine-rich repeat protein phosphatases (PHLPP) belong to the protein phosphatase magnesium/manganese-dependent family of Ser/Thr phosphatases. Their general role as tumor suppressors has been documented for over a decade. In recent years, accumulating evidence suggests that PHLPP isozymes have key regulatory roles in both innate and adaptive immunity. In macrophages, PHLPP1 dampens signaling through TLR4 and the IFN-γ receptor by altering cytosolic signaling pathways. Additionally, nuclear-localized PHLPP1 inhibits STAT1-mediated inflammatory gene expression by direct dephosphorylation at Ser 727. PHLPP1 also regulates the migratory and inflammatory capacity of neutrophils in vivo. Furthermore, PHLPP1-mediated dephosphorylation of AKT on Ser 473 is required for both the suppressive function of regulatory T cells and for the pro-apoptotic effects of PHLPP1 in B cell chronic lymphocytic leukemia. In the context of immune homeostasis, PHLPP1 expression is modulated in multiple cell types by inflammatory signals, and the dynamics of its expression have varying effects on the pathogenesis of inflammatory bowel disease and septic shock. In this review, we summarize recent findings on the functions of PHLPP in inflammatory and regulatory signaling in the context of both innate and adaptive immunity.

Protein kinase C fusion proteins are paradoxically loss of function in cancer.

Within the AGC kinase superfamily, gene fusions resulting from chromosomal rearrangements have been most frequently described for protein kinase C (PKC), with gene fragments encoding either the C-terminal catalytic domain or the N-terminal regulatory moiety fused to other genes. Kinase fusions that eliminate regulatory domains are typically gain of function and often oncogenic. However, several quality control pathways prevent accumulation of aberrant PKC, suggesting that PKC fusions may paradoxically be loss of function. To explore this topic, we used biochemical, cellular, and genome editing approaches to investigate the function of fusions that retain the portion of the gene encoding either the catalytic domain or regulatory domain of PKC. Overexpression studies revealed that PKC catalytic domain fusions were constitutively active but vulnerable to degradation. Genome editing of endogenous genes to generate a cancer-associated PKC fusion resulted in cells with detectable levels of fusion transcript but no detectable protein. Hence, PKC catalytic domain fusions are paradoxically loss of function as a result of their instability, preventing appreciable accumulation of protein in cells. Overexpression of a PKC regulatory domain fusion suppressed both basal and agonist-induced endogenous PKC activity, acting in a dominant-negative manner by competing for diacylglycerol. For both catalytic and regulatory domain fusions, the PKC component of the fusion proteins mediated the effects of the full-length fusions on the parameters examined, suggesting that the partner protein is dispensable in these contexts. Taken together, our findings reveal that PKC gene fusions are distinct from oncogenic fusions and present a mechanism by which loss of PKC function occurs in cancer.

Protein kinase Cα gain-of-function variant in Alzheimer's disease displays enhanced catalysis by a mechanism that evades down-regulation.

Conventional protein kinase C (PKC) family members are reversibly activated by binding to the second messengers Ca2+ and diacylglycerol, events that break autoinhibitory constraints to allow the enzyme to adopt an active, but degradation-sensitive, conformation. Perturbing these autoinhibitory constraints, resulting in protein destabilization, is one of many mechanisms by which PKC function is lost in cancer. Here, we address how a gain-of-function germline mutation in PKCα in Alzheimer's disease (AD) enhances signaling without increasing vulnerability to down-regulation. Biochemical analyses of purified protein demonstrate that this mutation results in an ∼30% increase in the catalytic rate of the activated enzyme, with no changes in the concentrations of Ca2+ or lipid required for half-maximal activation. Molecular dynamics simulations reveal that this mutation has both localized and allosteric effects, most notably decreasing the dynamics of the C-helix, a key determinant in the catalytic turnover of kinases. Consistent with this mutation not altering autoinhibitory constraints, live-cell imaging studies reveal that the basal signaling output of PKCα-M489V is unchanged. However, the mutant enzyme in cells displays increased sensitivity to an inhibitor that is ineffective toward scaffolded PKC, suggesting the altered dynamics of the kinase domain may influence protein interactions. Finally, we show that phosphorylation of a key PKC substrate, myristoylated alanine-rich C-kinase substrate, is increased in brains of CRISPR-Cas9 genome-edited mice containing the PKCα-M489V mutation. Our results unveil how an AD-associated mutation in PKCα permits enhanced agonist-dependent signaling via a mechanism that evades the cell's homeostatic down-regulation of constitutively active PKCα.

Critical role for cytosolic group IVA phospholipase A2 in early adipocyte differentiation and obesity.

Adipogenesis is the process of differentiation of immature mesenchymal stem cells into adipocytes. Elucidation of the mechanisms that regulate adipocyte differentiation is key for the development of novel therapies for the control of obesity and related comorbidities. Cytosolic group IVA phospholipase A2 (cPLA2α) is the pivotal enzyme in receptor-mediated arachidonic acid (AA) mobilization and attendant eicosanoid production. Using primary multipotent cells and cell lines predetermined to become adipocytes, we show here that cPLA2α displays a proadipogenic function that occurs very early in the adipogenic process. Interestingly, cPLA2α levels decrease during adipogenesis, but cPLA2α-deficient preadipocytes exhibit a reduced capacity to differentiate into adipocytes, which affects early and terminal adipogenic transcription factors. Additionally, the absence of the phospholipase alters proliferation and cell-cycle progression that takes place during adipogenesis. Preconditioning of preadipocytes with AA increases the adipogenic capacity of these cells. Moreover, animals deficient in cPLA2α show resistance to obesity when fed a high fat diet that parallels changes in the expression of adipogenic transcription factors of the adipose tissue. Collectively, these results show that preadipocyte cPLA2α activation is a hitherto unrecognized factor for adipogenesis in vitro and in vivo.

Lipin-1 integrates lipid synthesis with proinflammatory responses during TLR activation in macrophages.

Lipin-1 is a Mg(2+)-dependent phosphatidic acid phosphatase involved in the de novo synthesis of phospholipids and triglycerides. Using macrophages from lipin-1-deficient animals and human macrophages deficient in the enzyme, we show in this work that this phosphatase acts as a proinflammatory mediator during TLR signaling and during the development of in vivo inflammatory processes. After TLR4 stimulation lipin-1-deficient macrophages showed a decreased production of diacylglycerol and activation of MAPKs and AP-1. Consequently, the generation of proinflammatory cytokines like IL-6, IL-12, IL-23, or enzymes like inducible NO synthase and cyclooxygenase 2, was reduced. In addition, animals lacking lipin-1 had a faster recovery from endotoxin administration concomitant with a reduced production of harmful molecules in spleen and liver. These findings demonstrate an unanticipated role for lipin-1 as a mediator of macrophage proinflammatory activation and support a critical link between lipid biosynthesis and systemic inflammatory responses.

Enhanced activity of Alzheimer disease-associated variant of protein kinase Cα drives cognitive decline in a mouse model.

Exquisitely tuned activity of protein kinase C (PKC) isozymes is essential to maintaining cellular homeostasis. Whereas loss-of-function mutations are generally associated with cancer, gain-of-function variants in one isozyme, PKCα, are associated with Alzheimer's disease (AD). Here we show that the enhanced activity of one variant, PKCα M489V, is sufficient to rewire the brain phosphoproteome, drive synaptic degeneration, and impair cognition in a mouse model. This variant causes a modest 30% increase in catalytic activity without altering on/off activation dynamics or stability, underscoring that enhanced catalytic activity is sufficient to drive the biochemical, cellular, and ultimately cognitive effects observed. Analysis of hippocampal neurons from PKCα M489V mice reveals enhanced amyloid-ß-induced synaptic depression and reduced spine density compared to wild-type mice. Behavioral studies reveal that this mutation alone is sufficient to impair cognition, and, when coupled to a mouse model of AD, further accelerates cognitive decline. The druggability of protein kinases positions PKCα as a promising therapeutic target in AD.

Conventional protein kinase C in the brain: repurposing cancer drugs for neurodegenerative treatment?

Protein Kinase C (PKC) isozymes are tightly regulated kinases that transduce a myriad of signals from receptor-mediated hydrolysis of membrane phospholipids. They play an important role in brain physiology, and dysregulation of PKC activity is associated with neurodegeneration. Gain-of-function mutations in PKCα are associated with Alzheimer's disease (AD) and mutations in PKCγ cause spinocerebellar ataxia (SCA) type 14 (SCA14). This article presents an overview of the role of the conventional PKCα and PKCγ in neurodegeneration and proposes repurposing PKC inhibitors, which failed in clinical trials for cancer, for the treatment of neurodegenerative diseases.

The PHLPP1 N-Terminal Extension Is a Mitotic Cdk1 Substrate and Controls an Interactome Switch.

PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1) is a tumor suppressor that directly dephosphorylates a wide array of substrates, most notably the prosurvival kinase Akt. However, little is known about the molecular mechanisms governing PHLPP1 itself. Here, we report that PHLPP1 is dynamically regulated in a cell cycle-dependent manner and deletion of PHLPP1 results in mitotic delays and increased rates of chromosomal segregation errors. We show that PHLPP1 is hyperphosphorylated during mitosis by Cdk1 in a functionally uncharacterized region known as the PHLPP1 N-terminal extension (NTE). A proximity-dependent biotin identification (BioID) interaction screen revealed that during mitosis, PHLPP1 dissociates from plasma membrane scaffolds, such as Scribble, by a mechanism that depends on its NTE and gains proximity to kinetochore and mitotic spindle proteins such as KNL1 and TPX2. Our data are consistent with a model in which phosphorylation of PHLPP1 during mitosis regulates binding to its mitotic partners and allows accurate progression through mitosis. The finding that PHLPP1 binds mitotic proteins in a cell cycle- and phosphorylation-dependent manner may have relevance to its tumor-suppressive function.

mTORC2 controls the activity of PKC and Akt by phosphorylating a conserved TOR interaction motif.

The complex mTORC2 is accepted to be the kinase that controls the phosphorylation of the hydrophobic motif, a key regulatory switch for AGC kinases, although whether mTOR directly phosphorylates this motif remains controversial. Here, we identified an mTOR-mediated phosphorylation site that we termed the TOR interaction motif (TIM; F-x3-F-pT), which controls the phosphorylation of the hydrophobic motif of PKC and Akt and the activity of these kinases. The TIM is invariant in mTORC2-dependent AGC kinases, is evolutionarily conserved, and coevolved with mTORC2 components. Mutation of this motif in Akt1 and PKCßII abolished cellular kinase activity by impairing activation loop and hydrophobic motif phosphorylation. mTORC2 directly phosphorylated the PKC TIM in vitro, and this phosphorylation event was detected in mouse brain. Overexpression of PDK1 in mTORC2-deficient cells rescued hydrophobic motif phosphorylation of PKC and Akt by a mechanism dependent on their intrinsic catalytic activity, revealing that mTORC2 facilitates the PDK1 phosphorylation step, which, in turn, enables autophosphorylation. Structural analysis revealed that PKC homodimerization is driven by a TIM-containing helix, and biophysical proximity assays showed that newly synthesized, unphosphorylated PKC dimerizes in cells. Furthermore, disruption of the dimer interface by stapled peptides promoted hydrophobic motif phosphorylation. Our data support a model in which mTORC2 relieves nascent PKC dimerization through TIM phosphorylation, recruiting PDK1 to phosphorylate the activation loop and triggering intramolecular hydrophobic motif autophosphorylation. Identification of TIM phosphorylation and its role in the regulation of PKC provides the basis for AGC kinase regulation by mTORC2.

PHLPP1 counter-regulates STAT1-mediated inflammatory signaling.

Inflammation is an essential aspect of innate immunity but also contributes to diverse human diseases. Although much is known about the kinases that control inflammatory signaling, less is known about the opposing phosphatases. Here we report that deletion of the gene encoding PH domain Leucine-rich repeat Protein Phosphatase 1 (PHLPP1) protects mice from lethal lipopolysaccharide (LPS) challenge and live Escherichia coli infection. Investigation of PHLPP1 function in macrophages reveals that it controls the magnitude and duration of inflammatory signaling by dephosphorylating the transcription factor STAT1 on Ser727 to inhibit its activity, reduce its promoter residency, and reduce the expression of target genes involved in innate immunity and cytokine signaling. This previously undescribed function of PHLPP1 depends on a bipartite nuclear localization signal in its unique N-terminal extension. Our data support a model in which nuclear PHLPP1 dephosphorylates STAT1 to control the magnitude and duration of inflammatory signaling in macrophages.

The phosphatidic acid phosphatase lipin-1 facilitates inflammation-driven colon carcinogenesis.

Colon cancer is a devastating illness that is associated with gut inflammation. Here, we explored the possible role of lipin-1, a phosphatidic acid phosphatase, in the development of colitis-associated tumorigenesis. Azoxymethane and dextran sodium sulfate-treated (DSS-treated) animals deficient in lipin-1 harbored fewer tumors and carcinomas than WT animals due to decreased cellular proliferation, lower expression of antiapoptotic and protumorigenic factors, and a reduced infiltration of macrophages in colon tumors. They also displayed increased resistance to DSS-induced colitis by producing less proinflammatory cytokines and experiencing less immune infiltration. Lipin-1-deficient macrophages from the colon were less activated and displayed lower phosphatidic acid phosphatase activity than WT macrophages isolated from DSS-treated animals. Transference of WT macrophages into lipin-1-deficient animals was sufficient to increase colitis burden. Furthermore, treatment of lipin-1-deficient mice with IL-23 exacerbated colon inflammation. Analysis of human databases from colon cancer and ulcerative colitis patients showed that lipin-1 expression is increased in those disorders and correlates with the expression of the proinflammatory markers CXCL1 and CXCL2. And finally, clinically, LPIN1 expression had prognostic value in inflammatory and stem-cell subtypes of colon cancers. Collectively, these data demonstrate that lipin-1 is a critical regulator of intestinal inflammation and inflammation-driven colon cancer development.

Lipin-2 regulates NLRP3 inflammasome by affecting P2X7 receptor activation.

Mutations in human LPIN2 produce a disease known as Majeed syndrome, the clinical manifestations of which are ameliorated by strategies that block IL-1ß or its receptor. However the role of lipin-2 during IL-1ß production remains elusive. We show here that lipin-2 controls excessive IL-1ß formation in primary human and mouse macrophages by several mechanisms, including activation of the inflammasome NLRP3. Lipin-2 regulates MAPK activation, which mediates synthesis of pro-IL-1ß during inflammasome priming. Lipin-2 also inhibits the activation and sensitization of the purinergic receptor P2X7 and K+ efflux, apoptosis-associated speck-like protein with a CARD domain oligomerization, and caspase-1 processing, key events during inflammasome activation. Reduced levels of lipin-2 in macrophages lead to a decrease in cellular cholesterol levels. In fact, restoration of cholesterol concentrations in cells lacking lipin-2 decreases ion currents through the P2X7 receptor, and downstream events that drive IL-1ß production. Furthermore, lipin-2-deficient mice exhibit increased sensitivity to high lipopolysaccharide doses. Collectively, our results unveil lipin-2 as a critical player in the negative regulation of NLRP3 inflammasome.