Cancer-associated protein kinase C mutations reveal kinase's role as tumor suppressor.

Protein kinase C (PKC) isozymes have remained elusive cancer targets despite the unambiguous tumor promoting function of their potent ligands, phorbol esters, and the prevalence of their mutations. We analyzed 8% of PKC mutations identified in human cancers and found that, surprisingly, most were loss of function and none were activating. Loss-of-function mutations occurred in all PKC subgroups and impeded second-messenger binding, phosphorylation, or catalysis. Correction of a loss-of-function PKCß mutation by CRISPR-mediated genome editing in a patient-derived colon cancer cell line suppressed anchorage-independent growth and reduced tumor growth in a xenograft model. Hemizygous deletion promoted anchorage-independent growth, revealing that PKCß is haploinsufficient for tumor suppression. Several mutations were dominant negative, suppressing global PKC signaling output, and bioinformatic analysis suggested that PKC mutations cooperate with co-occurring mutations in cancer drivers. These data establish that PKC isozymes generally function as tumor suppressors, indicating that therapies should focus on restoring, not inhibiting, PKC activity.

Post-translational modification-centric base editor screens to assess phosphorylation site functionality in high throughput.

Signaling pathways that drive gene expression are typically depicted as having a dozen or so landmark phosphorylation and transcriptional events. In reality, thousands of dynamic post-translational modifications (PTMs) orchestrate nearly every cellular function, and we lack technologies to find causal links between these vast biochemical pathways and genetic circuits at scale. Here we describe the high-throughput, functional assessment of phosphorylation sites through the development of PTM-centric base editing coupled to phenotypic screens, directed by temporally resolved phosphoproteomics. Using T cell activation as a model, we observe hundreds of unstudied phosphorylation sites that modulate NFAT transcriptional activity. We identify the phosphorylation-mediated nuclear localization of PHLPP1, which promotes NFAT but inhibits NFκB activity. We also find that specific phosphosite mutants can alter gene expression in subtle yet distinct patterns, demonstrating the potential for fine-tuning transcriptional responses. Overall, base editor screening of PTM sites provides a powerful platform to dissect PTM function within signaling pathways.

Protein Kinase C Quality Control by Phosphatase PHLPP1 Unveils Loss-of-Function Mechanism in Cancer.

Protein kinase C (PKC) isozymes function as tumor suppressors in increasing contexts. In contrast to oncogenic kinases, whose function is acutely regulated by transient phosphorylation, PKC is constitutively phosphorylated following biosynthesis to yield a stable, autoinhibited enzyme that is reversibly activated by second messengers. Here, we report that the phosphatase PHLPP1 opposes PKC phosphorylation during maturation, leading to the degradation of aberrantly active species that do not become autoinhibited. Cancer-associated hotspot mutations in the pseudosubstrate of PKCß that impair autoinhibition result in dephosphorylated and unstable enzymes. Protein-level analysis reveals that PKCα is fully phosphorylated at the PHLPP site in over 5,000 patient tumors, with higher PKC levels correlating (1) inversely with PHLPP1 levels and (2) positively with improved survival in pancreatic adenocarcinoma. Thus, PHLPP1 provides a proofreading step that maintains the fidelity of PKC autoinhibition and reveals a prominent loss-of-function mechanism in cancer by suppressing the steady-state levels of PKC.

Protein kinase C: release from quarantine by mTORC2.

Protein kinase C (PKC) isozymes are maintained in a 'ready-to-go' but 'safe' autoinhibited conformation until second messenger binding unleashes an autoinhibitory pseudosubstrate to allow substrate phosphorylation. However, to gain this 'ready-to-go' conformation, PKC must be processed by a series of complex priming phosphorylations, the mechanism of which was enigmatic until now. Recent findings snapped the pieces of the phosphorylation puzzle into place to unveil a process that involves a newly described motif (TOR interaction motif, TIM), a well-described kinase [mechanistic target of rapamycin complex 2 (mTORC2)], and an often-used mechanism (autophosphorylation) to prime PKC to signal. This review highlights new insights into how phosphorylation controls PKC and discusses them in the context of common mechanisms for AGC kinase regulation by phosphorylation and autophosphorylation.

Cancer-associated mutations in protein kinase C theta are loss-of-function.

The Ca2+-independent, but diacylglycerol-regulated, novel protein kinase C (PKC) theta (θ) is highly expressed in hematopoietic cells where it participates in immune signaling and platelet function. Mounting evidence suggests that PKCθ may be involved in cancer, particularly blood cancers, breast cancer, and gastrointestinal stromal tumors, yet how to target this kinase (as an oncogene or as a tumor suppressor) has not been established. Here, we examine the effect of four cancer-associated mutations, R145H/C in the autoinhibitory pseudosubstrate, E161K in the regulatory C1A domain, and R635W in the regulatory C-terminal tail, on the cellular activity and stability of PKCθ. Live-cell imaging studies using the genetically-encoded fluorescence resonance energy transfer-based reporter for PKC activity, C kinase activity reporter 2 (CKAR2), revealed that the pseudosubstrate and C1A domain mutations impaired autoinhibition to increase basal signaling. This impaired autoinhibition resulted in decreased stability of the protein, consistent with the well-characterized behavior of Ca2+-regulated PKC isozymes wherein mutations that impair autoinhibition are paradoxically loss-of-function because the mutant protein is degraded. In marked contrast, the C-terminal tail mutation resulted in enhanced autoinhibition and enhanced stability. Thus, the examined mutations were loss-of-function by different mechanisms: mutations that impaired autoinhibition promoted the degradation of PKC, and those that enhanced autoinhibition stabilized an inactive PKC. Supporting a general loss-of-function of PKCθ in cancer, bioinformatics analysis revealed that protein levels of PKCθ are reduced in diverse cancers, including lung, renal, head and neck, and pancreatic. Our results reveal that PKCθ function is lost in cancer.

PHLPPing the Script: Emerging Roles of PHLPP Phosphatases in Cell Signaling.

Whereas protein kinases have been successfully targeted for a variety of diseases, protein phosphatases remain an underutilized therapeutic target, in part because of incomplete characterization of their effects on signaling networks. The pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP) is a relatively new player in the cell signaling field, and new roles in controlling the balance among cell survival, proliferation, and apoptosis are being increasingly identified. Originally characterized for its tumor-suppressive function in deactivating the prosurvival kinase Akt, PHLPP may have an opposing role in promoting survival, as recent evidence suggests. Additionally, identification of the transcription factor STAT1 as a substrate unveils a role for PHLPP as a critical mediator of transcriptional programs in cancer and the inflammatory response. This review summarizes the current knowledge of PHLPP as both a tumor suppressor and an oncogene and highlights emerging functions in regulating gene expression and the immune system. Understanding the context-dependent functions of PHLPP is essential for appropriate therapeutic intervention.

Protein kinase C showcases allosteric control: activation of LRRK1.

Allosteric regulation of multi-domain protein kinases provides a common mechanism to acutely control kinase activity. Protein kinase C serves as a paradigm for multi-domain proteins whose activity is exquisitely tuned by interdomain conformational changes that keep the enzyme off in the absence of appropriate stimuli, but unleash activity in response to second messenger binding. Allosteric regulation of protein kinase C signaling has been optimized not just for itself: Alessi and colleagues discover that protein kinase C phosphorylates LRRK1, a kinase with even more domains, at sites on its CORB GTPase domain to allosterically activate LRRK1.

Single-residue mutation in protein kinase C toggles between cancer and neurodegeneration.

Conventional protein kinase C (cPKC) isozymes tune the signaling output of cells, with loss-of-function somatic mutations associated with cancer and gain-of-function germline mutations identified in neurodegeneration. PKC with impaired autoinhibition is removed from the cell by quality-control mechanisms to prevent the accumulation of aberrantly active enzyme. Here, we examine how a highly conserved residue in the C1A domain of cPKC isozymes permits quality-control degradation when mutated to histidine in cancer (PKCß-R42H) and blocks down-regulation when mutated to proline in the neurodegenerative disease spinocerebellar ataxia (PKCγ-R41P). Using FRET-based biosensors, we determined that mutation of R42 to any residue, including lysine, resulted in reduced autoinhibition as indicated by higher basal activity and faster agonist-induced plasma membrane translocation. R42 is predicted to form a stabilizing salt bridge with E655 in the C-tail and mutation of E655, but not neighboring E657, also reduced autoinhibition. Western blot analysis revealed that whereas R42H had reduced stability, the R42P mutant was stable and insensitive to activator-induced ubiquitination and down-regulation, an effect previously observed by deletion of the entire C1A domain. Molecular dynamics (MD) simulations and analysis of stable regions of the domain using local spatial pattern (LSP) alignment suggested that P42 interacts with Q66 to impair mobility and conformation of one of the ligand-binding loops. Additional mutation of Q66 to the smaller asparagine (R42P/Q66N), to remove conformational constraints, restored degradation sensitivity. Our results unveil how disease-associated mutations of the same residue in the C1A domain can toggle between gain- or loss-of-function of PKC.

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.

mTOR Regulation of AGC Kinases: New Twist to an Old Tail.

The family of AGC kinases not only regulates cellular biology by phosphorylating substrates but is itself controlled by phosphorylation. Phosphorylation generally occurs at two conserved regions in these kinases: a loop near the entrance to the active site, termed the activation loop, that correctly aligns residues for catalysis, and a C-terminal tail whose phosphorylation at a site termed the hydrophobic motif stabilizes the active conformation. Whereas phosphorylation of the activation loop is well established to be catalyzed by the phosphoinositide-dependent kinase 1, the mechanism of phosphorylation of the C-tail hydrophobic motif has been controversial. For a subset of AGC kinases, which include most protein kinase C (PKC) isozymes and Akt, phosphorylation of the hydrophobic motif in cells was shown to depend on mTORC2 over 15 years ago, yet whether this was by direct phosphorylation or by another mechanism has remained elusive. The recent identification of a novel and evolutionarily conserved phosphorylation site on the C-tail, termed the TOR interaction motif (TIM), has finally unraveled the mystery of how mTORC2 regulates its client kinases. mTORC2 does not directly phosphorylate the hydrophobic motif; instead, it converts kinases such as PKC and Akt into a conformation that can ultimately autophosphorylate at the hydrophobic motif. Identification of the direct mTOR phosphorylation that facilitates autoregulation of the C-tail hydrophobic motif revises the activation mechanisms of mTOR-regulated AGC kinases. This new twist to an old tail opens avenues for therapeutic intervention. SIGNIFICANCE STATEMENT: The enzyme mTORC2 has been an enigmatic regulator of AGC kinases such as protein kinase C (PKC) and Akt. The recent discovery of a motif named the TOR interaction motif in the C-tail of these kinases solves the mystery: mTORC2 marks these kinases for maturity by, ultimately, facilitating autophosphorylation of another C-tail site, the hydrophobic motif.

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.

Both decreased and increased SRPK1 levels promote cancer by interfering with PHLPP-mediated dephosphorylation of Akt.

Akt activation is a hallmark of human cancers. Here, we report a critical mechanism for regulation of Akt activity by the splicing kinase SRPK1, a downstream Akt target for transducing growth signals to regulate splicing. Surprisingly, we find that SRPK1 has a tumor suppressor function because ablation of SRPK1 in mouse embryonic fibroblasts induces cell transformation. We link the phenotype to constitutive Akt activation from genome-wide phosphoproteomics analysis and discover that downregulated SRPK1 impairs the recruitment of the Akt phosphatase PHLPP1 (pleckstrin homology (PH) domain leucine-rich repeat protein phosphatase) to Akt. Interestingly, SRPK1 overexpression is also tumorigenic because excess SRPK1 squelches PHLPP1. Thus, aberrant SRPK1 expression in either direction induces constitutive Akt activation, providing a mechanistic basis for previous observations that SRPK1 is downregulated in some cancer contexts and upregulated in others.

PHLPPing the balance: restoration of protein kinase C in cancer.

Protein kinase signalling, which transduces external messages to mediate cellular growth and metabolism, is frequently deregulated in human disease, and specifically in cancer. As such, there are 77 kinase inhibitors currently approved for the treatment of human disease by the FDA. Due to their historical association as the receptors for the tumour-promoting phorbol esters, PKC isozymes were initially targeted as oncogenes in cancer. However, a meta-analysis of clinical trials with PKC inhibitors in combination with chemotherapy revealed that these treatments were not advantageous, and instead resulted in poorer outcomes and greater adverse effects. More recent studies suggest that instead of inhibiting PKC, therapies should aim to restore PKC function in cancer: cancer-associated PKC mutations are generally loss-of-function and high PKC protein is protective in many cancers, including most notably KRAS-driven cancers. These recent findings have reframed PKC as having a tumour suppressive function. This review focusses on a potential new mechanism of restoring PKC function in cancer - through targeting of its negative regulator, the Ser/Thr protein phosphatase PHLPP. This phosphatase regulates PKC steady-state levels by regulating the phosphorylation of a key site, the hydrophobic motif, whose phosphorylation is necessary for the stability of the enzyme. We also consider whether the phosphorylation of the potent oncogene KRAS provides a mechanism by which high PKC expression may be protective in KRAS-driven human cancers.

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.

Protein kinase C: perfectly balanced.

Protein kinase C (PKC) isozymes belong to a family of Ser/Thr kinases whose activity is governed by reversible release of an autoinhibitory pseudosubstrate. For conventional and novel isozymes, this is effected by binding the lipid second messenger, diacylglycerol, but for atypical PKC isozymes, this is effected by binding protein scaffolds. PKC shot into the limelight following the discovery in the 1980s that the diacylglycerol-sensitive isozymes are "receptors" for the potent tumor-promoting phorbol esters. This set in place a concept that PKC isozymes are oncoproteins. Yet three decades of cancer clinical trials targeting PKC with inhibitors failed and, in some cases, worsened patient outcome. Emerging evidence from cancer-associated mutations and protein expression levels provide a reason: PKC isozymes generally function as tumor suppressors and their activity should be restored, not inhibited, in cancer therapies. And whereas not enough activity is associated with cancer, variants with enhanced activity are associated with degenerative diseases such as Alzheimer's disease. This review describes the tightly controlled mechanisms that ensure PKC activity is perfectly balanced and what happens when these controls are deregulated. PKC isozymes serve as a paradigm for the wisdom of Confucius: "to go beyond is as wrong as to fall short."

Hypothesis: Unifying model of domain architecture for conventional and novel protein kinase C isozymes.

Protein kinase C (PKC) family members are multi-domain proteins whose function is exquisitely tuned by interdomain interactions that control the spatiotemporal dynamics of their signaling. Despite extensive mechanistic studies on this family of enzymes, no structure of a full-length enzyme that includes all domains has been solved. Here, we take into account the biochemical mechanisms that control autoinhibition, the properties of each individual domain, and previous structural studies to propose a unifying model for the general architecture of PKC family members. This model shows how the C2 domains of conventional and novel PKC isozymes, which have different topologies and different positions in the primary structure, can occupy the same position in the tertiary structure of the kinase. This common architecture of conventional and novel PKC isozymes provides a framework for understanding how disease-associated mutations impair PKC function.

Protein kinase C as a tumor suppressor.

Protein kinase C (PKC) has historically been considered an oncoprotein. This stems in large part from the discovery in the early 1980s that PKC is directly activated by tumor-promoting phorbol esters. Yet three decades of clinical trials using PKC inhibitors in cancer therapies not only failed, but in some cases worsened patient outcome. Why has targeting PKC in cancer eluded successful therapies? Recent studies looking at the disease for insight provide an explanation: cancer-associated mutations in PKC are generally loss-of-function (LOF), supporting an unexpected function as tumor suppressors. And, contrasting with LOF mutations in cancer, germline mutations that enhance the activity of some PKC isozymes are associated with degenerative diseases such as Alzheimer's disease. This review provides a background on the diverse mechanisms that ensure PKC is only active when, where, and for the appropriate duration needed and summarizes recent findings converging on a paradigm reversal: PKC family members generally function by suppressing, rather than promoting, survival signaling.

Genetic code expansion and live cell imaging reveal that Thr-308 phosphorylation is irreplaceable and sufficient for Akt1 activity.

The proto-oncogene Akt/protein kinase B (PKB) is a pivotal signal transducer for growth and survival. Growth factor stimulation leads to Akt phosphorylation at two regulatory sites (Thr-308 and Ser-473), acutely activating Akt signaling. Delineating the exact role of each regulatory site is, however, technically challenging and has remained elusive. Here, we used genetic code expansion to produce site-specifically phosphorylated Akt1 to dissect the contribution of each regulatory site to Akt1 activity. We achieved recombinant production of full-length Akt1 containing site-specific pThr and pSer residues for the first time. Our analysis of Akt1 site-specifically phosphorylated at either or both sites revealed that phosphorylation at both sites increases the apparent catalytic rate 1500-fold relative to unphosphorylated Akt1, an increase attributable primarily to phosphorylation at Thr-308. Live imaging of COS-7 cells confirmed that phosphorylation of Thr-308, but not Ser-473, is required for cellular activation of Akt. We found in vitro and in the cell that pThr-308 function cannot be mimicked with acidic residues, nor could unphosphorylated Thr-308 be mimicked by an Ala mutation. An Akt1 variant with pSer-308 achieved only partial enzymatic and cellular signaling activity, revealing a critical interaction between the γ-methyl group of pThr-308 and Cys-310 in the Akt1 active site. Thus, pThr-308 is necessary and sufficient to stimulate Akt signaling in cells, and the common use of phosphomimetics is not appropriate for studying the biology of Akt signaling. Our data also indicate that pThr-308 should be regarded as the primary diagnostic marker of Akt activity.