T m J0lrrur.u OF BIOLOOICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 269,No. 51,Issue of December 23,pp. 32358-32367, 1994 Printed in U.S.A.
Activation of Protein Kinase C Family Members by the Novel Polyphosphoinositides PtdIns-3,4-P2 and PtdIns-3,4,5-P3* (Received forpublication, June 14, 1994, and in revised form, September 26, 1994)
Alex TokerSP,Michael Meyern, K. Kishta Reddyll**, J. R. Falckll**, Rajindra AnejaSS, Sarla AnejaSS, Alejandro ParraSS, David J. Burnsn, Lawrence M. Ballasn, and Lewis C. Cantley4 From the $Department of Medicine, Division of Signal Dansduction, Beth Israel Hospital and the Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, llSphim Pharmaceuticals, Durham, North Carolina 27717, the IDepartment of Molecular Genetics, University of l’kxas Southwestern Medical Center, Dallas, Texas 75235, and the $$Functional Lipids Division, Langmuir Laboratory, Nutrimed Biotech, Cornel1 University Research Park, Ithaca, New York 14850
The effectof phosphoinositides on the activity of pro- or PtdIns-4,5-P2 to produce PtdIns-3-P, PtdIns-3,4P2, and Pttein kinase C (PKC) isotypes was investigated, PKC a, dIns-3,4,5-P3, respectively. PtdIns-3-P is present in unstimuPI, PII, y, 6, E, 4,and 5 were expressed in baculovirus- lated cells and does not significantly increase in response tocell infected insect cells and purified by column chromatog- activators (2). However, PtdIns-3,4-P2 and PtdIns-3,4,5-P3 are raphy. The calcium-activated PKC isotypes a, PI,PII, normally absent in unstimulated cells and appear withinsecand y were not significantly activated by any of the onds to minutes of cell activation. These lipids are not subphosphoinositides investigated (phosphatidylinosito1-4- strates of any known phospholipase type C and are therefore phosphate (PtdTns-4-P),PtdIns-3-P,PtdIns-4,5-P,, Pt- thought to be second messengers rather than precursors of dIns-3,4-P2,and PtdIns-3,4,5-P3) when added in thepres- second messengers (3). ence of concentrations of phosphatidylserine that give There is considerable circumstantial evidence that PtdInsmaximal stimulation. The calcium-insensitivePKC iso3,4-P, andor PtdIns-3,4,5-P3 act assecond messengers. Mutatypes 6, E, and 9 also showed little response to PtdInsoncoproteins that eliminate activationof 3-P, PtdIns4-P, or PtdIns-4,5-P2 whenthese lipids were tions in receptors and phosphoinositide 3-kinase have beenshown to compromise ceradded in the presence of phosphatidylserine. In contrast, PtdIns-3,4-PZand PtdIns-3,4,5-P3caused a 5-15- tain cellularresponses, including cell growth (11, chemotaxis (4, fold stimulation of these enzymes comparedwith phos- 5),membrane ruffling (4), and receptordown-regulation (6,7). phatidylserine alone. 50 % maximal stimulation of PKC E Inhibition of phosphoinositide 3-kinase with the fungal meof PDGFby PtdIns-3,4,5-P3occurred when this lipid was present taboliteWortmannincorrelateswithinhibition at about 1%of the carrier PtdIns-4,5-P2(about 100 m). dependent membraneruffling and cell growth: hormone secreThese lipids had little effect on baculovirus-expressed tion (91, insulin-induced glucose transport (101, and formyl pepPKC f which was constitutively active. A short chain tide-induced stimulation of neutrophils (11).Formyl peptideac- dependent actin filament rearrangement in neutrophils version of PtdIns-3,4,5-P3,dioctanoyl-PtdIns-3,4,5-P3, tivated PKC 6, E , and 4 in the absence of other lipids, temporally correlates with elevation of PtdIns-3,4,5-P3 (12,131. whereas a short chain version of PtdIns-4,5-P,, dihexA phosphatidylinositol 3-kinase in Saccharomyces cerevisiae anoyl-PtdIns-4,5-P2,did not. Since PtdIns-3,4-P2 and Pt- (Vps34) is critical for movement of luminal proteins from the dIns-3,4,5-P,are nominally absent in unstimulated cells Golgi to the vacuole (14, 15).However, this enzyme appears to and appear within seconds to minutes of stimulation by only phosphorylatePtdIns (16); PtdIns-3,4-P2 andPtdInsvarious cell activators, these lipids could act as second 3,4,5-P, have not been detected in yeast. Despite this strong messengers to activate PKC 6, E , or q in vivo. correlative evidence that lipid products of phosphoinositide 3-kinase are critical cellular signals, no in vivo targets for any of these lipids have beenidentified. Recently, PtdIns-3,4-P2 and Phosphoinositide 3-kinase is stimulated by a host of growth PtdIns-3,4,5-P3 werereported t o activatethe calcium-indefactors and other cellular activators(1).In vitro, this enzyme pendent, phorbol ester/DAG-insensitive PKC family member can phosphorylate phosphatidylinositol (PtdIns),’ PtdIns-4-P, PKC 6 in vitro,with no effect on a purified preparation of PKC from rat brain(17). These results were in part contradicted by * This work was supported by Research Grants GM41890 (to L. C. C. a separate report indicating thatPtdIns-3,4,5-P3 activated rat and A. T.)and GM37361 and GM49594 (to R. A,).The costs of publication of this article weredefrayedin part by the payment of page brain PKC more efficiently than did PtdIns-4,5-P2(18). PKC exists as a growing family of serinekhreonine kinases charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. with a wide range of physiological functions (reviewed in Ref. 5 To whom correspondence should be addressed: Dept. of Medicine, 19). To date, 12 members have beendescribed and subdivided Warren Alpert Building, Rm. 146, Beth Israel Hospital, 200 Longwood Ave., Boston, MA02115. Tel.: 617-278-3051; Fax: 617-278-3131; E-mail: into conventional PKC members comprising a, PI, PII, and y isoforms (activated by calcium, phospholipid, and DAG), novel
[email protected]. **Financially supported by the MizutaniFoundation for Glyco- PKCs comprising 6, E , ( L ) ,and B (insensitive tocalcium) and science. The abbreviations usedare: PtdIns, phosphatidylinositol; PKC, pronositol 3,4,5-trisphosphate;Ins-1,4,5-P8,inositol 1,4,5-trisphosphate; tein kinase C; PtdIns-3-P, phosphatidylinositol 3-phosphate; PtdInsSf9, Spodoptera fruIns-1,3,4,5-P4, inositol1,3,4,5-tetrakisphosphate; 4-P,phosphatidylinositol4-phosphate;PtdIns-4,5-P,,phosphatidyligiperda cells; ms, mass spectrometry;MBP, myelinbasic protein;PDGF, nositol4,5-bisphosphate;PtdIns-3,4-P2,phosphatidylinositol3,4-bisplatelet-derivedgrowth factor; DiC,PA, 1,2-dihexanoyl-sn-glycero-3phosphate;PtdIns-3,4,5-P3,phosphatidylinositol3,4,5-trisphosphate; PS, phosphatidylserine; PE, phosphatidylethanolamine;DAG, 1,2-di- phosphoric acid. B. C. Duckworth, L. Rameh, A. Kazlauskas, and L. C. Cantley, suboctanoyl-sn-glycerol; DiC,F‘tdIns-4,5-P2, dihexanoyl phosphatidylinosimitted for publication. to1 4,5-bisphosphate;DiC8PtdIns-3,4,5-P,, dioctanoylphosphatidyli-
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Activation of PKC by PtdIns-3,4,5-P3
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atypical PKCs comprising Jh and 6, a subgroup of PKC isoforms whose activity is not affected by phorbol ester or the natural activatorDAG (20-22). Yet another subgroup of PKCs may be defined by the recentlydescribed PKC p, with a potential signal peptide and transmembrane domain (23). Polyphosphoinositides such as PtdIns-4,5-P2 havebeen previously shown to activate PKC i n uitro (24-26), though these studies arecompromised by the factthat PtdIns-4,5-P2 can act both asa phospholipidcofactor and as an activator. It isunclear what role, if any, PtdIns-4,5-P2plays in the physiological actias arachidonic vation of PKC. Cis-unsaturated fatty acids such acid, oleic acid, linoleic acid, and linolenic acid have also been shown to stimulate the activity of PKC a, 0, y, E , and (20, 27-29). Of particular interest are recent reports that the potential second messenger, phosphatidic acid, is able to stimulate the activityof PKC q (30) and 6 (31). In an effort t o better understand the roIe of polyphosphoinositides in PKC activation, PKC a, PI, 011, y , 6, E , q, and 6 were expressed inbaculovirus-infected Sf9 insect cells and partially purified by column chromatography. The added advantage of little or no detectable PKC activity in wild-type insect cells provides a sourceof isoform pure PKC activity (32). In this study, we report the activationof PKC isoforms 6, E , and q by PtdIns-3,4-P2 and PtdIns-3,4,5-P3.Chemically synthesized short chain versions of PtdIns-3,4,5-P3 and PtdIns-4,5-P2 were used to test the specificity of the activation, Thefindings indicate thatof all isoforms tested, PKC E was themost sensitive to PtdIns-3,4,5-P3, whereas the activitiesof PKC a,PI, PII, and y were unaffected.
