Activation of PRK1 by phosphatidylinositol 4,5–bisphosphate and phosphatidylinositol 3,4,5–trisphosphate. A comparison with protein kinase C isotypes

Protein Phosphorylation Laboratory, Imperial Cancer Research Fund, London, United Kingdom.
Journal of Biological Chemistry (Impact Factor: 4.57). 10/1995; 270(38):22412-6. DOI: 10.1074/jbc.270.38.22412
Source: PubMed


As potential targets for polyphosphoinositides, activation of protein kinase C (PKC) isotypes (beta 1, epsilon, zeta, nu) and a member of the PKC-related kinase (PRK) family, PRK1, has been compared in vitro. PRK1 is shown to be activated by both phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2) as well as phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P3) either as pure sonicated lipids or in detergent mixed micelles. When presented as sonicated lipids, PtdIns-4,5-P2 and PtdIns-3,4,5-P3 were equipotent in activating PRK1, and, furthermore, sonicated phosphatidylinositol (PtdIns) and phosphatidylserine (PtdSer) were equally effective. In detergent mixed micelles, PtdIns-4,5-P2 and PtdIns-3,4,5-P3 also showed a similar potency, but PtdIns and PtdSer were 10-fold less effective in this assay. Similarly, PKC-beta 1, -epsilon, and -nu were all activated by PtdIns-4,5-P2 and PtdIns-3,4,5-P3 in detergent mixed micelles. The activation constants for PtdIns-4,5-P2 and PtdIns-3,4,5-P3 were essentially the same for all the kinases tested, implying no specificity in this in vitro analysis. Consistent with this conclusion, the effects of PtdIns-4,5-P2 and PtdIns-3,4,5-P3 were found to be inhibited at 10 mM Mg2+ and mimicked by high concentrations of inositol hexaphosphate and inositol hexasulfate. The similar responses of these two classes of lipid-activated protein kinase to these phosphoinositides are discussed in light of their potential roles as second messengers.

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    • "The lack of correlation between effector binding and function might either reflect the different conditions used for the functional and binding assays, or indirect effects of RhoA on effector function. The potential for such indirect effects is illustrated by the observations that GTP-bound RhoA binds both the myosin binding subunit (MBS), the regulatory subunit of myosin lightchain phosphatase, and ROCK-II, the kinase that phosphorylates MBS (Kimura et al., 1996); and that the association of RhoA with phosphatidylinositol 4-phosphate 5-kinase (PIP5K) (Ren et al., 1996) may also indirectly regulate PKN by altering PI-4,5–P2 concentrations (Palmer et al., 1995). However, in microinjection experiments we have been unable to block F39V-or Y42C-induced SRF activation by expression of RhoA.N19, a dominant interfering mutant (E.Sahai, unpublished data). "
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    ABSTRACT: The RhoA GTPase regulates diverse cellular processes including cytoskeletal reorganization, transcription and transformation. Although many different potential RhoA effectors have been identified, including two families of protein kinases, their roles in RhoA-regulated events remain unclear. We used a genetic screen to identify mutations at positions 37-42 in the RhoA effector loop that selectively disrupt effector binding, and used these to investigate the role of RhoA effectors in the formation of actin stress fibres, activation of transcription by serum response factor (SRF) and transformation. Interaction with the ROCK kinase and at least one other unidentified effector is required for stress fibre formation. Signalling to SRF by RhoA can occur in the absence of RhoA-induced cytoskeletal changes, and did not correlate with binding to any of the effectors tested, indicating that it may be mediated by an unknown effector. Binding to ROCK-I, but not activation of SRF, correlated with the activity of RhoA in transformation. The effector mutants should provide novel approaches for the functional study of RhoA and isolation of effector molecules involved in specific signalling processes.
    The EMBO Journal 04/1998; 17(5):1350-61. DOI:10.1093/emboj/17.5.1350 · 10.43 Impact Factor
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    • "Defekte auf diesem Weg können zu schweren Krankheiten wie Krebs oder M. Alzheimer führen. Phosphoinositide wirken als second messenger oder deren Vorläufer an der Signaltransduktion mit (Liscovitch und Cantley, 1995; Varsanyi et al., 1983, Vrolix et al., 1988; Bazenet et al., 1990; Starling et al., 1995; Gross et al., 1995; Palmer et al., 1995 "
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    ABSTRACT: Die PI4K230 ist eine Lipidkinase, deren Struktur auf Interaktionen mit anderen Proteinen und eine Rolle im Vesikeltransport hinweist. Lokalisation, Bedeutung der Aktivität und Einfluss der Domänen auf die Lokalisation und den intrazellulären Transport sowie die Kolokalisation mit Zellkompartimenten wurden untersucht. Eine Kinase-inaktive Mutante und N-terminale Deletionsmutanten wurden durch PCR-Mutagenese hergestellt. Wildtyp und Mutanten wurden in COS7-Zellen exprimiert und auf Lokalisation mittels Fluoreszenz am Konfokalmikroskop untersucht. Die Lokalisation von Zellkompartimenten und PI4K230 wurde verglichen. Die Lokalisation der PI4K230 ist unabhängig von der Aktivität. Das Helix-Loop-Helix-Motiv ist notwendig für die Lokalisation. Die PI4K230 kolokalisiert in einigen Fällen mit Lysosomen, Mitochondrien und Endoplasmatischem Retikulum. Überexprimierte PI4K230 tritt in Kompetition mit der endogenen PI4K230. Deletionsmutanten haben einen dominant- negativen Einfluss auf die PI4K230.
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    ABSTRACT: The research on 3T3-L1 adipocytes described in this thesis demonstrates how two different types of cellular stress inducing agents, namely the vicinal thiol binding agent arsenite and the conventional PKC-binding and -activating agent PMA act to increase glucose uptake in these cells. Whereas arsenite uses mainly the insulin-responsive GLUT4 transporter, PMA increases basal glucose transport through the GLUT1 transporter. As described in Chapter 3, arsenite-induced glucose uptake illustrates several requirements needed by any agent acting through GLUT4. These are, a tyrosine kinase activity, p38 MAPK activation and PKC-\lambda activity. Though PI-3' kinase activation is an essential step in insulin-signalling, this step is not required for arsenite-induced glucose uptake. Apparently, the need for tyrosine-kinase activity in arsenite induced glucose uptake resides in the ability to tyrosine-phosphorylate Cbl (see Chapter 3 Fig. 5). A further illustration of the importance of Cbl-tyrosine phosphorylation comes from our studies on rottlerin (Chapter 4). The ATP-depletion mediated by this pharmacological compound does not seem to be responsible for the observed inhibition of GLUT4 translocation (as was postulated by Kayali et al.[1]). Rather, aside from acting as an uncompetitive inhibitor of GLUT4, rottlerin hampers Cbl tyrosine phosphorylation, which leads to a 75% reduction in GLUT4 translocation (see Chapter 4, Fig. 3 and 4). Regrettably, the nature of the arsenite-induced tyrosine-kinase activity remains as of yet unidentified. Though the specific ability of arsenite to induce STAT5a tyrosine-phosphorylation in the mature adipocyte, should provide a straightforward tool to enable its identification (J.L González-Galindo, unpublished observations) Previously it had been demonstrated that insulin-induced p38 MAPK was involved in regulating the amount of glucose taken up by the cell without affecting GLUT4 translocation, suggesting some kind of intrinsic effect on the GLUT4 transporter itself [2]. Our observations on arsenite, a potent activator of p38 MAPK, illustrate a similar phenomenon in GLUT4-mediated stress-induced glucose uptake (see Chapter 3, Fig. 6). Subsequent research, described in a recently submitted manuscript, provides a detailed analysis of the involvement of p38 MAPK. These data demonstrate that p38 MAPK is involved in fine-tuning glucose uptake by regulating the turnover capacity of the GLUT4 transporter. A further note on the fine tuning of GLUT4-mediated glucose uptake comes from the observations on genistein, described in Chapter 5. This research suggests that in GLUT4 the turnover capacity for glucose can also be regulated through an intracellular ATP-binding Walker B motif akin to that described for GLUT1 [3]. Though further research is required to elucidate this mechanism, this theoretical resolution constitutes a significant step forwards towards understanding mechanisms in action after GLUT4 membrane translocation. If these observations are mechanistically linked in the cell remains to be elucidated. Aside from leading to enquiries into the mechanisms of insulin-induced glucose uptake, arsenite also opened up an avenue of more physiological research. We observed that arsenite-induced glucose uptake was sensitive to treatment with the insulin-resistance inducing agent dexamethasone. Subsequent analysis (described in Chapter 7) learned that although PI-3' kinase signalling is affected, in 3T3-L1 adipocytes the signalling pathway downstream is able to absorb this impediment. Rather, MKP-1 and -4 are upregulated in response to dexamethasone. Consequentially p38 MAPK activity is lost, leading to a reduction in glucose uptake. Given that MKP-4 is also upregulated in db/db- and ob/ob-mice [4], and that treatment of db/db mice with a glucocorticoid-receptor antagonist improves blood glucose levels [5;6], attenuation of p38 MAPK-signalling could be an important factor in type II diabetes. To enable the studies described in this chapter, a novel tool had to be developed. 3T3-L1 adipocytes have for long been inaccessible to ectopic expression of DNA. By the application of Lentivirus as described in Chapter 6, a large number of cells can be efficiently and reliably transduced. This novel tool will make the 3T3-L1 adipocyte readily amendable to routine molecular biological techniques, which will be of great benefit to the research field. In contrast to arsenite, PMA does not induce GLUT4 translocation, but acts solely through GLUT1. As illustrated in Chapter 8 of this thesis, in 3T3-L1 adipocytes the earliest and most PMA-sensitive PKC isoform is PKC-\betaII. But rather than activation, it is the concomitant degradation of this isoform which induces GLUT1 translocation. Further research (described in Chapter 9) highlighted the processes involved : First transcription of GLUT1, operating through the classical PKC-ERK-GLUT1 pathway. Second, translocation of GLUT1. This translocation is mediated by PKC-\lambda which is found associated with PKC-\betaII in the basal state. Thus upon degradation of the \betaII-isoform (or disruption of this complex by treatment with myristoylated pseudo-substrate peptides against \betaII) PKC-\lambda is released and free to act as a positional queue inducing translocation of the GLUT1 containing vesicles.
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