MALBON LABORATORY

CRAIG C. MALBON, PH.D., M.DIV., FAAAS, FRSM

Leading Professor 

Ph.D., Case Western Reserve University
Postdoctoral training: Brown University, Division of Biology & Medicine;
Department of Biology, Harvard University
Visiting Scholar, Princeton Theological Seminary
M. Div. (Ethics), Union Theological Seminary in New York City
Fellow, Center for Medical Humanities, Compassionate Care, & Bioethics
Department of Family, Population, & Preventive Medicine
631-444-3077  craig.malbon@stonybrook.edu

Signal Transduction in Differentiation and Development: Roles of Molecular Scaffold Molecules (e.g., AKAPs and Dishevelleds)

MALBON LABORATORY

Department of Pharmacology
Diabetes and Metabolic Diseases Research Center
School of Medicine
SUNY at Stony Brook, HSC-BST T7, Rm 156,
Stony Brook NY 11794-8651
Tel:631-444-7873 Fax:631-444-7696
e-mail: craig.malbon@stonybrook.edu

Malbon Laboratory Research

REGULATION OF HORMONE-SENSITIVE EFFECTOR SYSTEMS

The role of heterotrimeric G proteins in the signaling of Wnts has been established, from flies to mammals. Wnts signal, in part, via heptahelical G protein coupled receptors (GPCR) to effectors systems that control fundamental processes, such as adipogenesis, bone formation, cell progression, cell fate, and other aspects of early development. Dysregulation of Wnt signaling provides a basis for human disease, including cancer. The phosphoprotein Dishevelled (DVD) is essential to Wnt/b-

catenin (canonical), Wnt/ca²⁺/cGMP, and Wnt/planar cell polarity (PCP) pathways. Wnts promote mouse F9 embryonic teratocarcinoma cells (F9) to primitive endoderm (PE) and each of these effector pathways, via one/more or 3 mammalian Dvls. We propose 3 specific aims: (i) to define functional roles of mammalian Dvls on each major Wnt-stimulated pathway using siRNA and read outs of b-catenin stability, Lef/Tcf-sensitive transcription, Ca²⁺ imaging and cGMP analysis, JNK activation via Rho, and PE formation; (ii) to establish multivalency of Dvl interactions with GPCR, G proteins, kinases/phosphatases, and adaptor molecules critical to Wnt signaling making use of tandem affinity-based purification (TAP) and advanced proteomics (MALDI, QToF, and nanospray mass spectrometry), siRNA-based knock downs, enzyme inhibitors/dominant negative constructs, and eventual mutagenesis of potential docking sites; and (iii) to establish the functional consequences of spatial localization and trafficking of Dvl in naïve and Wnt-activated F9 cells, focusing on defining the dynamic aspects of activation /deactivation using fluorescence-based strategies (including e photon, fcs, and TIRF microscopy), energy transfer measurements in live cells (via BRET), and cell fractionation coupled with TAP-tagged Dvl followed by proteomics. The overarching hypothesis is that Dvls function as dynamic scaffolds for integration of cell signaling to a spectrum of diverse pathways, much like the AKAP protein family members function. Dvls both dock and are candidate substrates for many kinases/phosphatases, although the details of these interactive regulations remain to be established by proteomics. Real-time measurements of docking in live cells are made possible by BRET and parallel biochemical/proteomics assays that can define the status of Dvl with respect to protein phosphorylation. The functional basis for 3 mammalian Dvls, for their spatial and temporal trafficking, and for cell membrane association are addressed using in vivo and in vitro strategies. Understanding the multivalency and docking of Dvl open opportunity to intervene in the primary signaling pathways controlled by Wnts, including those whose dysregulation leads to cancer, birth defects, and altered bone formation. Dvls, as multivalent dynamic scaffolds, are high-value likely targets for new therapies.

STRUCTURE AND BIOLOGY OF BETA –ADRENERGIC RECEPTORS

A-Kinase Anchoring Proteins constitute a diverse family of scaffold proteins which share a common binding site for the RI/II subunit of PKA and are now recognized as scaffolds for multivalent cell signaling. AKAP250 (a.k.a., AKAP12, gravin, or a human homologue of SSeCKS) plays a critical role in signaling of the b2-adrenergic receptor (b2AR), the prototype for the superfamily of G-protein-coupled receptors (GPCR). We have demonstrated that AKAP250 is multivalent, providing ascaffold for b2AR signaling complexes that include minimally PKA, PKC, PP2B, Src, and the b2AR. Agonist activation of the b2AR stimulates eventual desensitization, sequestration, resensitization, and recycling of the receptor, a process disrupted in the absence of AKAP250. Recently we have mapped the domains of the b2AR as well as those for AKAP250 that provide the basis for protein-protein interactions

and a central role of protein phosphorylation in defining this dynamic interaction. The overarching goal of this research plan is to understand at the “meso”-scale, the dynamic role of AKAP scaffold in b2AR signaling complexes. Four specific aims target the goal: namely 1) to probe protein-lipid and protein-protein interactions of AKAP250 in b2AR signaling (focusing on three domains that may dictate scaffold-membrane association); 2) to identify new signaling molecules that constitute AKAP-based b2AR signaling complexes and to establish their function in the complex (making use of yeast-2-hybrid and HTS proteomic analysis of complex pull-downs); 3) to map the spatial organization and trafficking mechanisms of b2AR signaling complexes in response to stimulation by b-agonist (desensitization) and by insulin (counterregulation), using 2-photon confocal and bioluminescence resonance energy transfer (BRET) spectroscopy; and 4) to elucidate the ordered pattern of phosphorylation of key molecules constituting

AKAP-based b2AR signaling complexes and its function in the dynamic regulation of the complexes (using mass spectrometry in tandem with domain-specific mutagenesis). The targeted convergence of mass spectrometry-based proteomics, sensitive multi-photon confocal microscopy, BRET, and read-outs of localization, function, and protein-protein interactions create an unparalleled opportunity for success.

Atherosclerosis and Peripheral Apoprotein E Synthesis

The long term objective of this research is to understand the functional basis for the expression of lipoprotein (apo) E in peripheral tissues. ApoE is important for regulating systemic cholesterol transport metabolism. Among the plasma apolipoproteins, apoE is unusual in being expressed in many tissues and is involved in cellular processes and diseases that are independent of systemic lipoprotein metabolism. This proposal has two primary objectives. The first is to define the mechanisms by which low-levels of plasma apoE suppress atherosclerotic lesion development. Previous studies showed that

levels of transgenic apoE too low to correct hypercholesterolemia in apoE-deficient mice still blocked aortic lesion formation. The second goal is to determine how localized apoE expression in adrenocortical cells regulates the utilization of cholesterol for steroid production. These studies employ apoE-deficient mouse lines that have been engineered to express different levels of transgenic apoE selectively in the adrenal gland. The proposal has 3 aims. Aim 1 has 3 goals focused on how low-levels

(= 10-8M) of apoE alter the initial stages of lesion formation. Goal 1 will use quantitative real-time PCR to monitor expression of a set of candidate genes during initial stages of leukocyte recruitment to the vascular wall. Goal 2 will determine whether signaling pathways for platelet derived growth factor (PDGF) or those involving the transcription the transcription factors NF-aB and Egr-1 are activated in vascular cells at early stages, and whether low-level apoE alters this activation. Goal 3 will test which receptors of the LDL receptor family are important for the atheroprotective effects of low-level apoE. Airm 2 will use transgenic mice expressing apoE in adrenocortical cells to define mechanisms by which apoE alters cholesterol utilization. Goal 1 will use immunocytochemical approaches to evaluate the variegated pattern of transgenic apoE expression in the adrenal cortex and to test the effect of localized apoE on the expression of key proteins involved in the provision of substrate cholesterol to the steroidogenic pathway. Goal 2 will test which receptors of the LDL receptor family are important for the effects of apoE in adrenocortical cells. Aim 3 has collaborative projects to test the effects of low-level apoE. On neointimal formation after arterial injury and on regression of atherosclerotic lesions. These studies will provide new mechanistic formation about actions of apoE on cholesterol and metabolism and atherosclerosis.