Alexandra C. Newton

Protein Kinase C: Structure, Function, and Regulation

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 Cs 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, 2, 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.

Regulation of protein kinase C

Protein kinase C has been in the spotlight since the discovery two decades ago that it is activated by the lipid second messenger diacylglycerol. Despite protein kinase Cs enduring stage presence, the regulation and specific roles of its isozymes in defined cellular processes are still under intense investigation. Elucidation of the structures of protein kinase Cs regulatory modules, the discovery that phosphorylation regulates the enzyme, and the identification of targeting mechanisms have made the past year a significant one for unveiling how this ubiquitous class of enzymes operates.

Regulation of the ABC kinases by phosphorylation: protein kinase C as a paradigm

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.

Regulation of protein kinase C ζ by PI 3-kinase and PDK-1

BACKGROUND Protein kinase C zeta (PKC zeta) is a member of the PKC family of enzymes and is involved in a wide range of physiological processes including mitogenesis, protein synthesis, cell survival and transcriptional regulation. PKC zeta has received considerable attention recently as a target of phosphoinositide 3-kinase (PI 3-kinase), although the mechanism of PKC zeta activation is, as yet, unknown. Recent reports have also shown that the phosphoinositide-dependent protein kinase-1 (PDK-1), which binds with high affinity to the PI 3-kinase lipid product phosphatidylinositol-3,4,5-trisphosphate (Ptdins-3,4,5-P3), phosphorylates and potently activates two other PI 3-kinase targets, the protein kinases Akt/PKB and p70S6K. We therefore investigated whether PDK-1 is the kinase that activates PKC zeta. RESULTS In vivo, PI 3-kinase is both necessary and sufficient to activate PKC zeta. PDK-1 phosphorylates and activates PKC zeta in vivo, and we have shown that this is due to phosphorylation of threonine 410 in the PKC zeta activation loop. In vitro, PDK-1 phosphorylates and activates PKC zeta in a Ptdins-3,4,5-P3-enhanced manner. PKC zeta and PDK-1 are associated in vivo, and membrane targeting of PKC zeta renders it constitutively active in cells. CONCLUSIONS Our results have identified PDK-1 as the kinase that phosphorylates and activates PKC zeta in the PI 3-kinase signaling pathway. This phosphorylation and activation of PKC zeta by PDK-1 is enhanced in the presence of Ptdins-3,4-5-P3. Consistent with the notion that PKCs are enzymes that are regulated at the plasma membrane, a membrane-targeted PKC zeta is constitutively active in the absence of agonist stimulation. The association between PKC zeta and PDK-1 reveals extensive cross-talk between enzymes in the PI 3-kinase signaling pathway.

A genetically encoded fluorescent reporter reveals oscillatory phosphorylation by protein kinase C

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.

Protein kinase C is regulated in vivo by three functionally distinct phosphorylations

BACKGROUND Protein kinase Cs are a family of enzymes that transduce the plethora of signals promoting lipid hydrolysis. Here, we show that protein kinase C must first be processed by three distinct phosphorylations before it is competent to respond to second messengers. RESULTS We have identified the positions and functions of the in vivo phosphorylation sites of protein kinase C by mass spectrometry and peptide sequencing of native and phosphatase-treated kinase from the detergent-soluble fraction of cells. Specifically, the threonine at position 500 (T500) on the activation loop, and T641 and S660 on the carboxyl terminus of protein kinase C beta II are phosphorylated in vivo. T500 and S660 are selectively dephosphorylated in vitro by protein phosphatase 2A to yield an enzyme that is still capable of lipid-dependent activation, whereas all three residues are dephosphorylated by protein phosphatase 1 to yield an inactive enzyme. Biochemical analysis reveals that protein kinase C autophosphorylates on S660, that autophosphorylation on S660 follows T641 autophosphorylation, that autophosphorylation on S660 is accompanied by the release of protein kinase C into the cytosol, and that T500 is not an autophosphorylation site. CONCLUSIONS Structural and biochemical analyses of native and phosphatase-treated protein kinase C indicate that protein kinase C is processed by three phosphorylations. Firstly, trans-phosphorylation on the activation loop (T500) renders it catalytically competent to autophosphorylate. Secondly, a subsequent autophosphorylation on the carboxyl terminus (T641) maintains catalytic competence. Thirdly, a second autophosphorylation on the carboxyl terminus (S660) regulates the enzymes subcellular localization. The conservation of each of these residues (or an acidic residue) in conventional, novel and atypical protein kinase Cs underscores the essential role for each in regulating the protein kinase C family.

Cellular Signaling: Pivoting around PDK-1

the discovery that PDK-1 is the Akt/PKB upstream ki-Harvard Medical School nase came the observation that PDK-1 also phosphory-Boston, Massachusetts 02215 lates a number of other kinases, including p70S6-kinase † Department of Pharmacology (p70S6-K) and protein kinase C (PKC) (Figure 1) (for a relieves autoinhibition of the ac-hormones. Deregulation of this pathway is associated tive site, allowing PDK-1 to access Thr308 on the activa-with human diseases such as cancer and diabetes. The tion loop (Stokoe et al., 1997). Consistent with this, an importance of this pathway in cell biology is under-Akt/PKB mutant lacking the PH domain no longer re-scored by the fact that PI3K signaling influences both quires PtdIns-3,4,5-P 3 for PDK-1-mediated phosphory-cell survival and death, in addition to other fundamental lation in vitro and is constitutively phosphorylated and cellular functions such as growth, motility, differentia-active in cells (Filippa et al., 2000). Thus, the PH domain tion and insulin action. It does so by activating multiple masks the activation loop site and its release is required distinct secondary signaling cascades, and consider-for PDK-1 phosphorylation. able information exists about the precise biochemical Similarly, access of PDK-1 to the activation loop of mechanisms by which PI3K mediates these events. One PKC is conformationally regulated. In this case, the au-group of enzymes that has emerged as a key mediator toinhibitory pseudosubstrate sequence of PKC must be of the PI3K signal is the AGC superfamily of serine/ removed from the substrate binding cavity in order for threonine protein kinases (so named because it includes PDK-1 to phosphorylate PKC. The phosphorylation by protein kinases A, G, and C), long known to be critical PDK-1 of PRK/PKN is also conformationally regulated: components of the signal transduction machinery. Most these kinases require interaction with the small GTPase members of this family require an activating phosphory-Rho to induce a conformational change which enables lation, setting off the search for a potential upstream PDK-1 binding and phosphorylation at the activation kinase that was linked to the PI3K pathway. loop. In addition, binding of sphingosine to PAK has The search for such a kinase culminated with the recently been proposed to alter PAK in a manner that discovery in 1997 of a novel member of the AGC fam-permits phosphorylation of this kinase by PDK-1. Lastly, ily, the phosphoinositide-dependent kinase-1 (PDK-1). p70S6-K requires prior phosphorylation on its autoinhib-PDK-1 has now been shown to stand at a pivotal point in itory sequence …

The mammalian target of rapamycin complex 2 controls folding and stability of Akt and protein kinase C

The target of rapamycin (TOR), as part of the rapamycin‐sensitive TOR complex 1 (TORC1), regulates various aspects of protein synthesis. Whether TOR functions in this process as part of TORC2 remains to be elucidated. Here, we demonstrate that mTOR, SIN1 and rictor, components of mammalian (m)TORC2, are required for phosphorylation of Akt and conventional protein kinase C (PKC) at the turn motif (TM) site. This TORC2 function is growth factor independent and conserved from yeast to mammals. TM site phosphorylation facilitates carboxyl‐terminal folding and stabilizes newly synthesized Akt and PKC by interacting with conserved basic residues in the kinase domain. Without TM site phosphorylation, Akt becomes protected by the molecular chaperone Hsp90 from ubiquitination‐mediated proteasome degradation. Finally, we demonstrate that mTORC2 independently controls the Akt TM and HM sites in vivo and can directly phosphorylate both sites in vitro. Our studies uncover a novel function of the TOR pathway in regulating protein folding and stability, processes that are most likely linked to the functions of TOR in protein synthesis.

Protein kinase C: poised to signal

Nestled at the tip of a branch of the kinome, protein kinase C (PKC) family members are poised to transduce signals emanating from the cell surface. Cell membranes provide the platform for PKC function, supporting the maturation of PKC through phosphorylation, its allosteric activation by binding specific lipids, and, ultimately, promoting the downregulation of the enzyme. These regulatory mechanisms precisely control the level of signaling-competent PKC in the cell. Disruption of this regulation results in pathophysiological states, most notably cancer, where PKC levels are often grossly altered. This review introduces the PKC family and then focuses on recent advances in understanding the cellular regulation of its diacylglycerol-regulated members.

Regulation of conventional protein kinase C isozymes by phosphoinositide-dependent kinase 1 (PDK-1).

BACKGROUND Phosphorylation critically regulates the catalytic function of most members of the protein kinase superfamily. One such member, protein kinase C (PKC), contains two phosphorylation switches: a site on the activation loop that is phosphorylated by another kinase, and two autophosphorylation sites in the carboxyl terminus. For conventional PKC isozymes, the mature enzyme, which is present in the detergent-soluble fraction of cells, is quantitatively phosphorylated at the carboxy-terminal sites but only partially phosphorylated on the activation loop. RESULTS This study identifies the recently discovered phosphoinositide-dependent kinase 1, PDK-1, as a regulator of the activation loop of conventional PKC isozymes. First, studies in vivo revealed that PDK-1 controls the amount of mature (carboxy-terminally phosphorylated) conventional PKC. More specifically, co-expression of the conventional PKC isoform PKC betaII with a catalytically inactive form of PDK-1 in COS-7 cells resulted in both the accumulation of non-phosphorylated PKC and a corresponding decrease in PKC activity. Second, studies in vitro using purified proteins established that PDK-1 specifically phosphorylates the activation loop of PKC alpha and betaII. The phosphorylation of the mature PKC enzyme did not modulate its basal activity or its maximal cofactor-dependent activity. Rather, the phosphorylation of non-phosphorylated enzyme by PDK-1 triggered carboxy-terminal phosphorylation of PKC, thus providing the first step in the generation of catalytically competent (mature) enzyme. CONCLUSIONS We have shown that PDK-1 controls the phosphorylation of conventional PKC isozymes in vivo. Studies performed in vitro establish that PDK-1 directly phosphorylates PKC on the activation loop, thereby allowing carboxy-terminal phosphorylation of PKC. These data suggest that phosphorylation of the activation loop by PDK-1 provides the first step in the processing of conventional PKC isozymes by phosphorylation.