SUMMARY 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 PKCβ 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 PKCβ 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.

, followed by location-specific diacylglycerol generation. In response to UTP, phosphorylation of GolgiCKAR was sustained the longest, driven by the persistence of DAG, whereas phosphorylation of CytoCKAR was of the shortest duration, driven by high phosphatase activity. Our data reveal that the magnitude and duration of PKC signaling is location-specific and controlled by the level of phosphatase activity and persistence of DAG at each location.Cells respond dynamically to environmental cues conveyed by complex networks of signal transduction. Phosphorylation is the archetypal language that relays information from environmental stimuli throughout the cell; thus, hundreds of kinases and phosphatases exist to regulate the phosphorylation status of intracellular substrates. A delicate balance of phosphorylation and dephosphorylation underlies cellular decisions ranging from controlling global functions, such as proliferation or apoptosis, to regulating specialized functions, such as secretion of hormones (2, 3). Disturbing this balance can lead to disease states, most notably cancer (4).The protein kinase C (PKC) 2 family of Ser/Thr kinases transduces an abundance of extracellular signals that control diverse cellular functions, including differentiation, memory, and apoptosis. There are 10 mammalian isozymes of the PKC family, and they share a conserved COOH-terminal kinase core as well as an NH 2 -terminal autoinhibitory pseudosubstrate peptide that is lodged in the active site under resting conditions. PKC isoforms are classified into three subcategories (conventional, novel, and atypical) based on differing composition of their regulatory modules, which lie between the kinase core and inhibitory pseudosubstrate peptide (5). Conventional isoforms of PKC (cPKCs; ␣, ␤I, ␤II, and ␥) contain a tandem C1 repeat followed by a C2 domain, which allow them to respond to the second messengers diacylglycerol (DAG) and Ca 2ϩ , respectively. When extracellular signals stimulate phosphoinositide hydrolysis, DAG is produced and Ca 2ϩ is released. The binding of these second messengers to the regulatory domains results in translocation of cPKCs to cellular membranes. Both second messengers must be present for high affinity membrane binding, an event that provides the energy to disengage the inhibitory pseudosubstrate peptide from the active site, allowing downstream signaling (6). Novel isoforms of PKC (nPKCs; ␦, ⑀, , and ) are similarly activated by membrane binding; however, the novel C2 domain of the nPKCs cannot bind Ca 2ϩ . For these isozymes, high affinity membrane binding is achieved exclusively by the C1 domain, which compensates by having an increased affinity for DAG (7). Consequently, this subclass is regulated by DAG production but not by Ca 2ϩ release. Atypical PKCs and are unique in that they are not regulated by either DAG or Ca 2ϩ ; their regulatory region consists of an atypical C1 domain that does not bind DAG and a PB1 (Phox and Bem 1) domain, recently recognized for its role in protein-protein interactions ...

The C1 domain mediates the diacylglycerol (DAG)-dependent translocation of conventional and novel protein kinase C (PKC) isoforms. In novel PKC isoforms (nPKCs), this domain binds membranes with sufficiently high affinity to recruit nPKCs to membranes in the absence of any other targeting mechanism. In conventional PKC (cPKC) isoforms, however, the affinity of the C1 domain for DAG is two orders of magnitude lower, necessitating the coordinated binding of the C1 domain and a Ca 2؉ -regulated C2 domain for translocation and activation. Here we identify a single residue that tunes the affinity of the C1b domain for DAG-(but not phorbol ester-) containing membranes. This residue is invariant as Tyr in the C1b domain of cPKCs and invariant as Trp in all other PKC C1 domains. Binding studies using model membranes, as well as live cell imaging studies of yellow fluorescent protein-tagged C1 domains, reveal that Trp versus Tyr toggles the C1 domain between a species with sufficiently high affinity to respond to agonist-produced DAG to one that is unable to respond to physiological levels of DAG. In addition, we show that while Tyr at this switch position causes cytosolic localization of the C1 domain under unstimulated conditions, Trp targets these domains to the Golgi, likely due to basal levels of DAG at this region. Thus, Trp versus Tyr at this key position in the C1 domain controls both the membrane affinity and localization of PKC. The finding that a single residue controls the affinity of the C1 domain for DAG-containing membranes provides a molecular explanation for why 1) DAG alone is sufficient to activate nPKCs but not cPKCs and 2) nPKCs target to the Golgi. Protein kinase C (PKC)3 is a critical transducer of intracellular signaling pathways, with a variety of outputs, most notably tumor promotion (1, 2). The hallmark of PKC activation is its translocation to membranes (3). This translocation is mediated through the ligand-dependent engagement of two membranetargeting modules: the C1 (ligand: diacylglycerol or DAG) and C2 (ligand: Ca 2ϩ ) domains. The regulatory domains vary within the PKC family, which is subdivided into three groups based on regulation. The conventional PKC isoforms (cPKCs: ␣, ␤I/II, ␥) contain two tandem C1 domains and one conventional C2 domain; consequently, cPKCs are regulated both by DAG and Ca 2ϩ . The novel isoforms (nPKCs: ␦, ⑀, , and ) contain two tandem C1 domains and a non-Ca 2ϩ /membrane-binding novel C2 domain; consequently, nPKCs are regulated only by DAG. Atypical PKCs (aPKCs: and ) contain a single non-DAGbinding ("atypical") C1 domain and no C2 domain; as a result, aPKCs are regulated by neither DAG nor Ca 2ϩ (4). The C1 domain is an ϳ8-kDa domain that binds DAG or the potent DAG-mimicking phorbol esters, such as phorbol-12-myristate-13-acetate (PMA) and phorbol dibutyrate (PDBu) (5). Structural studies have established that all C1 domains have a similar fold (6 -11). An "unzipped" ␤ sheet forms a groove lined by hydrophobic residues in which membrane-embedded diacylg...

SUMMARY Dynamic actin cytoskeletal reorganization is integral to cell motility. Profilins are well-characterized regulators of actin polymerization; however, functional differences among co-expressed profilin isoforms are not well defined. Here, we demonstrate that profilin-1 and profilin-2 differentially regulate membrane protrusion, motility, and invasion; these processes are promoted by profilin-1 and suppressed by profilin-2. Compared to profilin-1, profilin-2 preferentially drives actin polymerization by the Ena/VASP protein, EVL. Profilin-2 and EVL suppress protrusive activity and cell motility by an actomyosin contractility-dependent mechanism. Importantly, EVL or profilin-2 downregulation enhances invasion in vitro and in vivo. In human breast cancer, lower EVL expression correlates with high invasiveness and poor patient outcome. We propose that profilin-2/EVL-mediated actin polymerization enhances actin bundling and suppresses breast cancer cell invasion.

SummaryThe lipid second messenger diacylglycerol (DAG) controls the rate, amplitude, duration, and location of protein kinase C (PKC) activity in the cell. There are three classes of PKC isozymes and, of these, the conventional and novel isozymes are acutely controlled by DAG. The kinetics of DAG production at various intracellular membranes, the intrinsic affinity of specific isoforms for DAG-containing membranes, the coordinated use of additional membrane-binding modules, the intramolecular regulation of DAG sensitivity, and the competition from other DAG-responsive proteins together result in a unique, context-dependent activation signature for each isoform. This review focuses on the spatiotemporal dynamics of PKC activation and how it is controlled by lipid second messengers.2008 IUBMB IUBMB Life, 60(12): 782-789, 2008