Many proteins associated with the plasma membrane are known to partition into submicroscopic sphingolipid- and cholesterol-rich domains called lipid rafts, but the determinants dictating this segregation of proteins in the membrane are poorly understood. We suppressed the tendency of Aequorea fluorescent proteins to dimerize and targeted these variants to the plasma membrane using several different types of lipid anchors. Fluorescence resonance energy transfer measurements in living cells revealed that acyl but not prenyl modifications promote clustering in lipid rafts. Thus the nature of the lipid anchor on a protein is sufficient to determine submicroscopic localization within the plasma membrane.

The protein kinase C family of enzymes transduces the myriad of signals promoting lipid hydrolysis. The prevalence of this enzyme family in signaling is exemplified by the diverse transduction mechanisms that result in the generation of protein kinase C's activator, diacylglycerol. Signals that stimulate members of the large families of G protein-coupled receptors, tyrosine kinase receptors, or non-receptor tyrosine kinases can cause diacylglycerol production, either rapidly by activation of specific phospholipase Cs or more slowly by activation of phospholipase D to yield phosphatidic acid and then diacylglycerol (1-3). In addition, fatty acid generation by phospholipase A2 activation modulates protein kinase C activity (3). Thus, multiple receptor pathways feeding into multiple lipid pathways have the common end result of activating protein kinase C by production of its second messenger.Phorbol esters, potent tumor promoters, can substitute for diacylglycerol in activating protein kinase C (1-3). Unlike diacylglycerol, phorbol esters are not readily metabolized, and treatment of cells with these molecules results in prolonged activation of protein kinase C. As a result, phorbol esters have proved invaluable in dissecting out protein kinase C-catalyzed phosphorylations in vivo.In addition to regulation by diacylglycerol or phorbol esters, all isozymes of protein kinase C require phosphatidylserine, an acidic lipid located exclusively on the cytoplasmic face of membranes, and some isozymes require Ca 2ϩ for optimal activity (4 -7). This review discusses the structure of the protein kinase C family, its enzymatic function, and how structure and function are regulated by 1) cofactors and 2) phosphorylation. StructureMembers of the protein kinase C family are a single polypeptide, comprised of an N-terminal regulatory region (approximately 20 -40 kDa) and a C-terminal catalytic region (approximately 45 kDa) (Fig. 1). Cloning of the first isozymes in the mid-1980s revealed four conserved domains: C1-C4 (8). Each is a functional module, and many unrelated proteins have one or the other (9). The function of each of these domains has been established by extensive biochemical and mutational analysis; the C1 domain contains a Cys-rich motif, duplicated in most isozymes, that forms the diacylglycerol/phorbol ester binding site (Fig. 1, orange) (7); this domain is immediately preceded by an autoinhibitory pseudosubstrate sequence ( Fig. 1, green) (10); the C2 domain contains the recognition site for acidic lipids and, in some isozymes, the Ca 2ϩ -binding site (Fig. 1, yellow) (9). The C3 and C4 domains form the ATP-and substrate-binding lobes of the kinase core (Fig. 1, pink and cyan) (11). The regulatory and catalytic halves are separated by a hinge region that becomes proteolytically labile when the enzyme is membrane-bound (6); the proteolytically generated kinase domain (protein kinase M), freed of inhibition by the pseudosubstrate, is constitutively active (12).To date, 11 protein kinase C isozymes have been identifie...

Akt/protein kinase B critically regulates the balance between cell survival and apoptosis. Phosphorylation of Akt at two key sites, the activation loop and the hydrophobic motif, activates the kinase and promotes cell survival. The mechanism of dephosphorylation and signal termination is unknown. Here, we identify a protein phosphatase, PH domain leucine-rich repeat protein phosphatase (PHLPP), that specifically dephosphorylates the hydrophobic motif of Akt (Ser473 in Akt1), triggering apoptosis and suppressing tumor growth. The effects of PHLPP on apoptosis are prevented in cells expressing an S473D construct of Akt, revealing that the hydrophobic motif is the primary cellular target of PHLPP. PHLPP levels are markedly reduced in several colon cancer and glioblastoma cell lines that have elevated Akt phosphorylation. Reintroduction of PHLPP into a glioblastoma cell line causes a dramatic suppression of tumor growth. These data are consistent with PHLPP terminating Akt signaling by directly dephosphorylating and inactivating Akt.

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Phosphorylation plays a central role in regulating the activation and signalling lifetime of protein kinases A, B (also known as Akt) and C. These kinases share three conserved phosphorylation motifs: the activation loop segment, the turn motif and the hydrophobic motif. This review focuses on how phosphorylation at each of these sites regulates the maturation, signalling and down-regulation of PKC as a paradigm for how these sites control the function of the ABC kinases.

Akt/protein kinase B controls cell growth, proliferation, and survival. We recently discovered a novel phosphatase PHLPP, for PH domain leucine-rich repeat protein phosphatase, which terminates Akt signaling by directly dephosphorylating and inactivating Akt. Here we describe a second family member, PHLPP2, which also inactivates Akt, inhibits cell-cycle progression, and promotes apoptosis. These phosphatases control the amplitude of Akt signaling: depletion of either isoform increases the magnitude of agonist-evoked Akt phosphorylation by almost two orders of magnitude. Although PHLPP1 and PHLPP2 both dephosphorylate the same residue (hydrophobic phosphorylation motif) on Akt, they differentially terminate Akt signaling by regulating distinct Akt isoforms. Knockdown studies reveal that PHLPP1 specifically modulates the phosphorylation of HDM2 and GSK-3alpha through Akt2, whereas PHLPP2 specifically modulates the phosphorylation of p27 through Akt3. Our data unveil a mechanism to selectively terminate Akt-signaling pathways through the differential inactivation of specific Akt isoforms by specific PHLPP isoforms.

Signals transduced by kinases depend on the extent and duration of substrate phosphorylation. We generated genetically encoded fluorescent reporters for PKC activity that reversibly respond to stimuli activating PKC. Specifically, phosphorylation of the reporter expressed in mammalian cells causes changes in fluorescence resonance energy transfer (FRET), allowing real time imaging of phosphorylation resulting from PKC activation. Targeting of the reporter to the plasma membrane, where PKC is activated, reveals oscillatory phosphorylation in HeLa cells in response to histamine. Each oscillation in substrate phosphorylation follows a calcium oscillation with a lag of ∼10 s. Novel FRET-based reporters for PKC translocation, phosphoinositide bisphosphate conversion to IP3, and diacylglycerol show that in HeLa cells the oscillatory phosphorylations correlate with Ca2+-controlled translocation of conventional PKC to the membrane without oscillations of PLC activity or diacylglycerol. However, in MDCK cells stimulated with ATP, PLC and diacylglycerol fluctuate together with Ca2+ and phosphorylation. Thus, specificity of PKC signaling depends on the local second messenger-controlled equilibrium between kinase and phosphatase activities to result in strict calcium-controlled temporal regulation of substrate phosphorylation.