The National Institute of Diabetes & Digestive & Kidney Diseases

This laboratory is investigating molecular processes required for establishing a terminally differentiated organism from a homogeneous population of totipotent cells and is defining signal transduction pathways that specify developmental cell fates and pattern formation. By characterizing receptor-mediated cascades and nuclear targets, our research serves to identify mechanisms that are basic to multicellular differentiation.

Organizing the embryonic body plan is a major requisite governing early metazoan development, with morphogen signaling central to establish and specify cell fates. One of these pathways involves Wnt/wingless signaling and includes 7-transmembrane (7-TM) target receptors and a downstream effector, the protein kinase GSK3. In Dictyostelium, stimulation of a family of 7-TM receptors by its ligand, the secreted chemoattractant/morphogen cAMP, also establishes a fundamental developmental organization, the anterior/posterior axis. We have shown that different cAMP receptor subtypes act antagonistically to promote or to inhibit cell-specific differentiation and axis formation. These antagonistic pathways converge at GSK3 in the context of cell fate determination. The latter inhibitory pathway parallels Wnt/wingless signalling through the 7-span Frizzled receptors to control GSK3 activity and axes formation in Drosophila, Xenopus, and other metazoa. Separately, we have identified a novel tyrosine kinase, ZAK1, that is required for receptor-mediated activation of GSK3 during development and have shown that recombinant ZAK1 will phosphorylate and consequently activate mammalian GSK3 in vitro. Remarkably, this 7-TM receptor-dependent activation of GSK3 may act independently of G proteins. These studies may reveal new mechanisms involving protein tyrosine kinases that are critical for cell fate specification or tumor suppression in other systems.

Other receptor-mediated pathways in Dictyostelium have an absolute requirement for G protein signaling. During early development, a pulsatile release of cAMP directs chemotatic migration and induces gene expression. The cAMP signal is transduced through the membrane by a receptor/G protein coupled pathway that regulates adenylyl cyclase (AC). AC is transiently activated by a cAMP signal, but rapidly adapts to a persistent cAMP signal. While G-beta is implicated in AC activation, the mechanism for adaptation of the response to cAMP has been unknown. We have identified a novel G-alpha (Ga9) in Dictyostelium that may participate in the adaptation pathway and the Ga9-nulls are hypersensitive to the newly identified secreted factor APF that potentiates the chemotatic response to cAMP. APF was purified to homogeneity and two proteins were individually isolated and are being sequenced. These observations suggest that Ga9 is part of an inhibitory signaling network that directs cell movement and senses cell density.

We also have a very strong interest in understanding how signaling circuits ultimately regulate cell-specific patterns of gene expression. We have analyzed several developmentally regulated promoters and have purified CRTF, a novel Zn-finger transcription factor, isolated its gene, and created null strains. CRTF is required for early development, and data suggest that CRTF activity is dependent upon G protein-signaling through the cAMP receptors. Studies are in progress to understand structure/function relationships of CRTF activation with regard to developmentally regulated gene expression.

Finally, we maintain an active collaboration to understand molecular mechanisms required for lipogenic and lipolytic action in mammalian adiopocytes. Primary focus has centered on the lipid droplet-associated proteins Perilipin and ADRP and on HSL, the hormone sensitive lipase, using knock-out and transgenic mouse models.

 
Multiple signaling pathways that ultimately regulate chemotaxis can be attenuated by a single heterotrimeric Ga protein in Dictyostelium discoideum. Shown here is a composite image of Dictyostelium cells moving toward a pipette(gray)that is emitting a chemoattractant. Relative to wild-type cells (blue), ga9-null cells (green) are
hyperpolarized and rarely produce lateral pseudopods, indicative of a loss of a negative regulator of chemotaxis. Cells expressing constitutively activated Ga9 (red) display the expected opposite phenotype, are poorly polarized, and produce numerous lateral pseudopods. In the background, a population of ga9-null cells chemotax to form aggregation centers during development. (For details, see Brzostowski et al., Genes Dev. 2004 18: 805-815).

1. Rosel D, Kimmel AR The COP9 signalosome regulates cell proliferation of Dictyostelium discoideum. Eur J Cell Biol , 2006. [Full Text/Abstract]

2. Khurana T, Brzostowski JA, Kimmel AR A Rab21/LIM-only/CH-LIM complex regulates phagocytosis via both activating and inhibitory mechanisms. EMBO J (24): 2254-64, 2005. [Full Text/Abstract]

3. Naude B, Brzostowski JA, Kimmel AR, Wellems TE Dictyostelium discoideum expresses a malaria chloroquine resistance mechanism upon transfection with mutant, but not wild-type, Plasmodium falciparum transporter PfCRT. J Biol Chem (280): 25596-603, 2005. [Full Text/Abstract]

4. Xu G, Sztalryd C, Lu X, Tansey JT, Gan J, Dorward H, Kimmel AR, Londos C Post-translational regulation of adipose differentiation-related protein by the ubiquitin/proteasome pathway. J Biol Chem (280): 42841-7, 2005. [Full Text/Abstract]

5. Liu X, Rubin JS, Kimmel AR Rapid, Wnt-induced changes in GSK3beta associations that regulate beta-catenin stabilization are mediated by Galpha proteins. Curr Biol (15): 1989-97, 2005. [Full Text/Abstract]

6. Brzostowski JA, Parent CA, Kimmel AR A G alpha-dependent pathway that antagonizes multiple chemoattractant responses that regulate directional cell movement. Genes Dev (18): 805-15, 2004. [Full Text/Abstract]

7. Faix J, Kreppel L, Shaulsky G, Schleicher M, Kimmel AR A rapid and efficient method to generate multiple gene disruptions in Dictyostelium discoideum using a single selectable marker and the Cre-loxP system. Nucleic Acids Res (32): e143, 2004. [Full Text/Abstract]

8. Kimmel AR, Firtel RA Breaking symmetries: regulation of Dictyostelium development through chemoattractant and morphogen signal-response. Curr Opin Genet Dev (14): 540-9, 2004. [Full Text/Abstract]

9. Kreppel L, Fey P, Gaudet P, Just E, Kibbe WA, Chisholm RL, Kimmel AR dictyBase: a new Dictyostelium discoideum genome database. Nucleic Acids Res (32): D332-3, 2004. [Full Text/Abstract]

10. Kimmel AR Parent CA Gough NR Spatial and Temporal Dynamics of Signaling Components Involved in the Control of Chemotaxis in Dictyostelium discoideum. Sci STKE (2004): TR3, 2004. [Full Text/Abstract]

11. Hickenbottom SJ Kimmel AR Londos C Hurley JH Structure of a Lipid Droplet Protein; The PAT Family Member TIP47. Structure (Camb) (12): 1199-207, 2004. [Full Text/Abstract]

12. Kimmel AR, Parent CA, Gough NR Teaching resources. Spatial and temporal dynamics of signaling components involved in the control of chemotaxis in Dictyostelium discoideum. Sci STKE (2004): tr3, 2004. [Full Text/Abstract]

13. Tansey JT, Huml AM, Vogt R, Davis KE, Jones JM, Fraser KA, Brasaemle DL, Kimmel AR, Londos C Functional studies on native and mutated forms of perilipins. A role in protein kinase A-mediated lipolysis of triacylglycerols. J Biol Chem (278): 8401-6, 2003. [Full Text/Abstract]

14. Su CL, Sztalryd C, Contreras JA, Holm C, Kimmel AR, Londos C Mutational analysis of the hormone-sensitive lipase translocation reaction in adipocytes. J Biol Chem (278): 43615-9, 2003. [Full Text/Abstract]

15. Sztalryd C, Xu G, Dorward H, Tansey JT, Contreras JA, Kimmel AR, Londos C Perilipin A is essential for the translocation of hormone-sensitive lipase during lipolytic activation. J Cell Biol (161): 1093-103, 2003. [Full Text/Abstract]

16. Kimmel AR, Parent CA The signal to move: D. discoideum go orienteering. Science (300): 1525-7, 2003. [Full Text/Abstract]

17. Miura S Gan JW Brzostowski J Parisi MJ Schultz CJ Londos C Oliver B Kimmel AR Functional conservation for lipid storage droplet association among Perilipin, ADRP, and TIP47 (PAT)-related proteins in mammals, Drosophila, and Dictyostelium. J Biol Chem (277): 32253-7, 2002. [Full Text/Abstract]

18. Brzostowski JA Johnson C Kimmel AR Galpha-mediated inhibition of developmental signal response. Curr Biol (12): 1199-208, 2002. [Full Text/Abstract]

19. Brzostowski JA, Johnson C, Kimmel AR Galpha-mediated inhibition of developmental signal response. Curr Biol (12): 1199-208, 2002. [Full Text/Abstract]

20. Kreppel L, Kimmel AR Genomic database resources for Dictyostelium discoideum. Nucleic Acids Res (30): 84-6, 2002. [Full Text/Abstract]

21. Kim L Harwood A Kimmel AR Receptor-dependent and tyrosine phosphatase-mediated inhibition of GSK3 regulates cell fate choice. Dev Cell(3): 523-32, 2002. [Full Text/Abstract]

22. Lu X Gruia-Gray J Copeland NG Gilbert DJ Jenkins NA Londos C Kimmel AR The murine perilipin gene: the lipid droplet-associated perilipins derive from tissue-specific, mRNA splice variants and define a gene family of ancient origin. Mamm Genome (12): 741-9, 2001. [Full Text/Abstract]

23. Kim L Kimmel AR GSK3, a master switch regulating cell-fate specification and tumorigenesis. Curr Opin Genet Dev(10): 508-14, 2000. [Full Text/Abstract]

24. Kim L Liu J Kimmel AR The novel tyrosine kinase ZAK1 activates GSK3 to direct cell fate specification. Cell(99): 399-408, 1999. [Full Text/Abstract]

25. Balint-Kurti P Ginsburg GT Liu J Kimmel AR Non-autonomous regulation of a graded, PKA-mediated transcriptional activation signal for cell patterning. Development (125): 3947-54, 1998. [Full Text/Abstract]

26. Mu X Lee B Louis JM Kimmel AR Sequence-specific protein interaction with a transcriptional enhancer involved in the autoregulated expression of cAMP receptor 1 in Dictyostelium. Development (125): 3689-98, 1998. [Full Text/Abstract]

27. Jin T Soede RD Liu J Kimmel AR Devreotes PN Schaap P Temperature-sensitive Gbeta mutants discriminate between G protein-dependent and -independent signaling mediated by serpentine receptors. EMBO J (17): 5076-84, 1998. [Full Text/Abstract]

28. Ginsburg GT Kimmel AR Autonomous and nonautonomous regulation of axis formation by antagonistic signaling via 7-span cAMP receptors and GSK3 in Dictyostelium. Genes Dev (11): 2112-23, 1997. [Full Text/Abstract]