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10 th PBL in calcium- and phospholipid signaling May 3-14, 2010 Md. Shahidul Islam, M.D., Ph.D.

10 th PBL in calcium- and phospholipid signaling May 3-14, 2010 Md. Shahidul Islam, M.D., Ph.D. Karolinska Instititet, Institutionen för klinisk forskning och utbildning, Forekningscentrum Södersjukhuset Shaisl@ki.se. cis. epi. allo. myo. myo. muco. neo. D-chiro(+). L-chiro(-).

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10 th PBL in calcium- and phospholipid signaling May 3-14, 2010 Md. Shahidul Islam, M.D., Ph.D.

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  1. 10 th PBL in calcium- and phospholipid signaling May 3-14, 2010 Md. Shahidul Islam, M.D., Ph.D. Karolinska Instititet, Institutionen för klinisk forskning och utbildning, Forekningscentrum Södersjukhuset Shaisl@ki.se

  2. cis epi allo myo myo muco neo D-chiro(+) L-chiro(-) scyllo The Nine Inositol Isomers

  3. Diacylglycerol kinase CDP-Diacylglycerol Phosphatidylinositol Inositol CMP

  4. InsP InsP2 InsP3 PI PIP PIP2 CDP-DG PA DG

  5. Inositol 1,4,5 trisphosphate

  6. Phospholipase A1 O Phospholipase A2 R H2C O C O R C O CH O H2C O P O X Phospholipase C Phospholipase D Cleavage sites of phospholipases

  7. Measurement of Inositol Phospholipid Metabolism • 32Pi incorporation into phospholipids • 3[H]inositol phosphate accummulation • Separation of 3[H]inositol phosphates by Dowex anion exchange chromatography • Measurement of mass of Ins(1,4,5)P3 by radio receptor assay

  8. Measurement of 3[H]inositol phosphate accumulation • Use inositol-free medium • (RPMI contain 190 mM inositol) • Use inositol-free serum (FBS 550 mM; Horse serum 200 mM inositol) • Add carrier inositol • No antibiotics • Label for prolonged period • Use Lithium • Inhibits Ins-1-P/Ins(1,3,4)P3 phosphatase and inositol monophasphatase

  9. Ins(1,4,5)P3 3-Kinase 5-Phosphatase Ins(1,4)P2 Ins(1,3,4,5)P4 *inhibited by lithium Ins(1,3,4)P3 *Ins(1,4)P2 /Ins(1,3,4)P3-1 phosphatase 3-phosphatase; 4-phosphatase *Inositol monophosphatase Inositol

  10. On switch Off switch cAMP cAMP PDEs Adenelyl cyclase Ins(1,4,5)P3 5-Phosphatase 3-Kinase

  11. Islam ms et al 1991, FEBS lett. 288:27

  12. Members of PI-PLC superfamily • Four mammalian PI-PLC b (b1-b4) • Two PI-PLCg (g1 and g2) • Four PI-PLC d (d1-d4) • PI-PLC e • PLCp21 and PLCnorpA are drosophila homolog of PLCb • PI-PLC b regulated by G-Proteins • PI-PLC g regulated by tyrosine kinases • PI-PLCd regulated by Ca2+ • PI-PLCe activated by GTP-Ras

  13. Pleckstrin/PH domain • Pleckstrin: platelet protein; substrate of PKC • PH domain: about 100 amino acid module • PH domain containing proteins PI-PLC Many KInases GTPases GTPase activating proteins Nucleotide exchange factors • Many PH containing proteins interact with G-proteins and membrane phospholipids

  14. SH2 Domain • About 100 aa domains: homology to the sequences found in nonreceptor tyrosine kinases of src family • Respond to tyrosine phosphorylation by binding to the phosphorylated sequences • On protein-protein interaction through SH2 domains, one of the protein may be relocalized

  15. SH3 domain • 60-85 aa stretches • Frequently occur together with SH2 domain • Involved in interaction with proteins containing proline-rich sequences • SH3 domain on PLCg may associate with actin network

  16. PH X X X Y Y Y PLCb N C PH PH SH2 PH SH3 PLCg N C PH PLCd N C Domain structure of PI-PLC family

  17. Activation of PI-PLC b by a subunit of G protein • alpha subunits of Gq family: Gq, G11, G14-16 • bind to C terminal part of PI-PLC: G-box • activate mainly PI-PLC b1 and b3

  18. Regulation of PI-PLC • PI-PLC b by G proteins • PI-PLC g by tyrosine kinase receptors • PI-PLC d by Ca2+ ?

  19. Activation of PI-PLC b by bg subunit of G protein • Gbg released from G-proteins, specially from G1 • Binds at the N terminal part of PI-PLC (PH domain) • Activates mainly PI-PLC b2 and b3

  20. PI-PLC g is activated by tyrosine phosphorylation • Tyrosine kinase receptors e.g. PDGF • Ligand binding, receptor dimerization • Mutual transphosphorylation of tyrosines on the receptor • SH2 domain of PI-PLCg docks on to the phosphorylated tyrosines • PI-PLCg is phosphorylated on tyrosine residues and this activates PI-PLC

  21. Activation of PI-PLCg requires two events • Association with the tyrosine phosphorylated receptor through SH2 domain • Phosphorylation of specific tyrosine residues on the PI-PLCg

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