W Todd Miller

Stony Brook University, Stony Brook, New York, United States

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Publications (74)462.44 Total impact

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    ABSTRACT: Podoplanin (PDPN) is a transmembrane glycoprotein that promotes tumor cell migration, invasion, and cancer metastasis. In fact, PDPN expression is induced in many types of cancer. Thus, PDPN has emerged as a functionally relevant cancer biomarker and chemotherapeutic target. PDPN contains 2 intracellular serine residues that are conserved between species ranging from mouse to humans. Recent studies indicate that protein kinase A (PKA) can phosphorylate PDPN in order to inhibit cell migration. However, the number and identification of specific residues phosphorylated by PKA have not been defined. In addition, roles of other kinases that may phosphorylate PDPN to control cell migration have not been investigated. We report here that cyclin dependent kinase 5 (CDK5) can phosphorylate PDPN in addition to PKA. Moreover, results from this study indicate that PKA and CDK5 cooperate to phosphorylate PDPN on both intracellular serine residues to decrease cell motility. These results provide new insight into PDPN phosphorylation dynamics and the role of PDPN in cell motility. Understanding novel mechanisms of PDPN intracellular signaling could assist with designing novel targeted chemotherapeutic agents and procedures. Copyright © 2015. Published by Elsevier Inc.
    Experimental Cell Research 05/2015; DOI:10.1016/j.yexcr.2015.04.019 · 3.37 Impact Factor
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    ABSTRACT: Despite significant evidence to the contrary, the view that phosphatases are ″non-specific″ still pervades the field. Systems biology approaches to defining how signal transduction pathways are integrated at the level of whole organisms also often downplay the contribution of phosphatases, defining them as ″erasers″ that serve merely to restore the system to its basal state. Here, we present a study that counteracts the idea of ″nonspecific phosphatases″. We have characterized two structurally similar and functionally related kinases, BRK and SRC, which are regulated by combinations of activating autophosphorylation and inhibitory, C-terminal sites of tyrosine phosphorylation. We demonstrated specificity at the level of the kinases in that SRMS phosphorylated the C-terminus of BRK, but not SRC; in contrast, CSK is the kinase responsible for C-terminal phosphorylation of SRC, but not BRK. For the phosphatases, we observed that RNAi-mediated suppression of PTP1B resulted in opposing effects on the activity of BRK and SRC, and have defined the mechanisms underlying this specificity. PTP1B inhibited BRK by directly dephosphorylating the Tyr-342 autophosphorylation site. In contrast, PTP1B potentiated SRC activity, but not by dephosphorylating SRC itself directly; instead, PTP1B regulated the interaction between CBP/PAG and CSK. SRC associated with, and phosphorylated, the transmembrane protein CBP/PAG at Tyr-317, resulting in CSK recruitment. We identified PAG as a substrate of PTP1B, and dephosphorylation abolished recruitment of the inhibitory kinase CSK. Overall, these findings illustrate how the combinatorial effects of PTKs and PTPs may be integrated to regulate signaling, with both classes of enzymes displaying exquisite specificity. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 04/2015; DOI:10.1074/jbc.M115.651703 · 4.60 Impact Factor
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    ABSTRACT: The insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R) are highly related receptor tyrosine kinases with a disulfide-linked homodimeric architecture. Ligand binding to the receptor ectodomain triggers tyrosine autophosphorylation of the cytoplasmic domains, which stimulates catalytic activity and creates recruitment sites for downstream signalling proteins. Whether the two phosphorylated tyrosine kinase domains within the receptor dimer function independently or cooperatively to phosphorylate protein substrates is not known. Here we provide crystallographic, biophysical and biochemical evidence demonstrating that the phosphorylated kinase domains of IR and IGF1R form a specific dimeric arrangement involving an exchange of the juxtamembrane region proximal to the kinase domain. In this dimer, the active position of α-helix C in the kinase N lobe is stabilized, which promotes downstream substrate phosphorylation. These studies afford a novel strategy for the design of small-molecule IR agonists as potential therapeutic agents for type 2 diabetes.
    Nature Communications 03/2015; 6:6406. DOI:10.1038/ncomms7406 · 10.74 Impact Factor
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    Noriko Yokoyama, W Todd Miller
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    Noriko Yokoyama, W Todd Miller
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    ABSTRACT: Anaplastic lymphoma kinase (ALK) is a member of the receptor tyrosine kinase superfamily. The ALK gene is a site of frequent mutation and chromosomal rearrangement in various types of human cancers. A novel chromosomal translocation was recently identified in human colorectal cancer between the ALK gene and chromosome 2, open reading frame 44 (C2orf44), a gene of unknown function. As a first step in understanding the oncogenic properties of this fusion protein, C2orf44 cDNA was cloned and the encoded protein was characterized, which was designated as WD repeat and coiled coil containing protein (WDCP). A C-terminal proline-rich segment in WDCP was shown to mediate binding to the Src homology 3 domain of the Src family kinase hematopoietic cell kinase (Hck). Co-expression with Hck lead to tyrosine phosphorylation of WDCP. Chromatographic fractionation of WDCP-containing lysates indicates that the protein exists as an oligomer in mammalian cells. These results suggest that, in the context of the ALK-C2orf44 gene fusion, WDCP imposes an oligomeric structure on ALK that results in constitutive kinase activation and signaling.
    01/2015; 3(1):9-13. DOI:10.3892/br.2014.374
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    Stevan R Hubbard, W Todd Miller
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    ABSTRACT: When insulin-like growth factor-1 (IGF1) binds to its receptor, a physical constraint is released that allows the two transmembrane helices to come together to facilitate activation of the receptor.
    eLife Sciences 10/2014; 3. DOI:10.7554/eLife.04909 · 8.52 Impact Factor
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    ABSTRACT: The development of the phosphotyrosine-based signaling system predated the evolution of multicellular animals. Single-celled choanoflagellates, the closest living relatives to metazoans, possess numerous tyrosine kinases, including Src family nonreceptor tyrosine kinases. Choanoflagellates also have Csk (C-terminal Src kinase), the enzyme that regulates Src in metazoans; however, choanoflagellate Csk kinases fail to repress the cognate Src. Here, we have cloned and characterized Src and Csk kinases from Ministeria vibrans, a filasterean (the sister group to metazoans and choanoflagellates). The two Src kinases (MvSrc1 and MvSrc2) are enzymatically active Src kinases, although they have low activity towards mammalian cellular proteins. Unexpectedly, MvSrc2 has significant Ser/Thr kinase activity. The Csk homolog (MvCsk) is enzymatically inactive, and fails to repress MvSrc activity. We suggest that the low activity of MvCsk is due to sequences in the SH2/kinase interface, and we show that a point mutation in this region partially restores MvCsk activity. The inactivity of filasterean Csk kinases is consistent with a model in which the stringent regulation of Src family kinases arose more recently in evolution, after the split between choanoflagellates and multicellular animals.
    Biochemistry 02/2014; 53(8). DOI:10.1021/bi4016499 · 3.19 Impact Factor
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    ABSTRACT: Podoplanin (PDPN) is a transmembrane receptor that affects the activities of RHO, ezrin, and other proteins to promote tumor cell motility, invasion, and metastasis. PDPN is found in many types of cancer and may serve as a tumor biomarker and chemotherapeutic target. The intracellular region of PDPN contains only two serines, and these are conserved in mammals including mice and humans. We generated cells from the embryos of homozygous null PDPN knockout mice to investigate the relevance of these serines to cell growth and migration on a clear (PDPN free) background. We report here that one or both of these serines can be phosphorylated by PKA (protein kinase A). We also report that conversion of these serines to nonphosphorylatable alanine residues enhances cell migration, while their conversion to phosphomimetic aspartate residues decreases cell migration. These results indicate that PKA can phosphorylate PDPN to decrease cell migration. In addition, we report that PDPN expression in fibroblasts causes them to facilitate the motility and viability of neighboring melanoma cells in coculture. These findings shed new light on how PDPN promotes cell motility, its role in tumorigenesis, and its utility as a functionally relevant biomarker and chemotherapeutic target.
    Journal of Biological Chemistry 03/2013; DOI:10.1074/jbc.C112.446823 · 4.60 Impact Factor
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    ABSTRACT: Background Receptor tyrosine kinases (RTKs) are crucial components of signal transduction systems in multicellular animals. Surprisingly, numerous RTKs have been identified in the genomes of unicellular choanoflagellates and other protists. Here, we report the first biochemical study of a unicellular RTK, namely RTKB2 from Monosiga brevicollis. Results We cloned, expressed, and purified the RTKB2 kinase, and showed that it is enzymatically active. The activity of RTKB2 is controlled by autophosphorylation, as in metazoan RTKs. RTKB2 possesses six copies of a unique domain (designated RM2) in its C-terminal tail. An isolated RM2 domain (or a synthetic peptide derived from the RM2 sequence) served as a substrate for RTKB2 kinase. When phosphorylated, the RM2 domain bound to the Src homology 2 domain of MbSrc1 from M. brevicollis. NMR structural studies of the RM2 domain indicated that it is disordered in solution. Conclusions Our results are consistent with a model in which RTKB2 activation stimulates receptor autophosphorylation within the RM2 domains. This leads to recruitment of Src-like kinases (and potentially other M. brevicollis proteins) and further phosphorylation, which may serve to increase or dampen downstream signals. Thus, crucial features of signal transduction circuitry were established prior to the evolution of metazoans from their unicellular ancestors.
    BMC Biochemistry 02/2013; 14(1):4. DOI:10.1186/1471-2091-14-4 · 1.94 Impact Factor
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    ABSTRACT: Phosphotyrosine-based signaling plays a vital role in cellular communication in multicellular organisms. Unexpectedly, unicellular choanoflagellates (the closest phylogenetic group to metazoans) possess numbers of tyrosine kinases that are comparable to those in complex metazoans. Here, we have characterized tyrosine kinases from the filasterean Capsaspora owczarzaki, a unicellular protist representing the sister group to choanoflagellates and metazoans. Two Src-like tyrosine kinases have been identified in C. owczarzaki (CoSrc1 and CoSrc2), both of which have the arrangement of SH3, SH2, and catalytic domains seen in mammalian Src kinases. In Capsaspora cells, CoSrc1 and CoSrc2 localize to punctate structures in filopodia that may represent primordial focal adhesions. We have cloned, expressed, and purified both enzymes. CoSrc1 and CoSrc2 are active tyrosine kinases. Mammalian Src kinases are normally regulated in a reciprocal fashion by autophosphorylation in the activation loop (which increases activity) and by Csk-mediated phosphorylation of the C-terminal tail (which inhibits activity). Similar to mammalian Src kinases, the enzymatic activities of CoSrc1 and CoSrc2 are increased by autophosphorylation in the activation loop. We have identified a Csk-like kinase (CoCsk) in the genome of C. owczarzaki. We cloned, expressed, and purified CoCsk and found that it has no measurable tyrosine kinase activity. Furthermore, CoCsk does not phosphorylate or regulate CoSrc1 or CoSrc2 in cells or in vitro, and CoSrc1 and CoSrc2 are active in Capsaspora cell lysates. Thus, the function of Csk as a negative regulator of Src family kinases appears to have arisen with the emergence of metazoans.
    Biochemistry 10/2012; 51(41):8267–8277. DOI:10.1021/bi300965h · 3.19 Impact Factor
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    Barbara P Craddock, W Todd Miller
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    ABSTRACT: The insulin-like growth factor I receptor (IGF1R) is overexpressed in several forms of human cancer, and it has emerged as an important target for anticancer drug design. Cancer genome sequencing efforts have recently identified three somatic mutations in IGF1R: A1374V, a deletion of S1278 in the C-terminal tail region of the receptor, and M1255I in the C-terminal lobe of the kinase catalytic domain. The possible effects of these mutations on IGF1R activity and biological function have not previously been tested. Here, we tested the effects of the mutations on the in vitro biochemical activity of IGF1R and on major IGF1R signaling pathways in mammalian cells. While the mutations do not affect the intrinsic tyrosine kinase activity of the receptor, we demonstrate that the basal (unstimulated) levels of MAP kinase and Akt activation are increased in the mutants (relative to wild-type IGF1R). We hypothesize that the enhanced signaling potential of these mutants is due to changes in protein-protein interactions between the IGF1R C-terminus and cellular substrates or modulators.
    06/2012; 2012:804801. DOI:10.1155/2012/804801
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    ABSTRACT: The role of Src in tumorigenesis has been extensively studied since the work of Peyton Rous over a hundred years ago. Src is a non-receptor tyrosine kinase that plays key roles in signaling pathways controlling tumor cell growth and migration. Src regulates the activities of numerous molecules to induce cell transformation. However, transformed cells do not always migrate and realize their tumorigenic potential. They can be normalized by surrounding nontransformed cells by a process called contact normalization. Tumor cells need to override contact normalization to become malignant or metastatic. In this review, we discuss the role of Src in cell migration and contact normalization, with emphasis on Cas and Abl pathways. This paradigm illuminates several chemotherapeutic targets and may lead to the identification of new biomarkers and the development of effective anticancer treatments.
    Genes & cancer 05/2012; 3(5-6):426-35. DOI:10.1177/1947601912458583
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    W Todd Miller
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    ABSTRACT: Tyrosine phosphorylation is an essential element of signal transduction in multicellular animals. Although tyrosine kinases were originally regarded as specific to the metazoan lineage, it is now clear that they evolved prior to the split between unicellular and multicellular eukaryotes (≈600million years ago). Genome analyses of choanoflagellates and other protists show an abundance of tyrosine kinases that rivals the most complex animals. Some of these kinases are orthologs of metazoan enzymes (e.g., Src), but others display unique domain compositions not seen in any metazoan. Biochemical experiments have highlighted similarities and differences between the unicellular and multicellular tyrosine kinases. In particular, it appears that the complex systems of kinase autoregulation may have evolved later in the metazoan lineage.
    Biochimica et Biophysica Acta 03/2012; 1823(6):1053-7. DOI:10.1016/j.bbamcr.2012.03.009 · 4.66 Impact Factor
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    ABSTRACT: Choanoflagellates are considered to be the closest living unicellular relatives of metazoans. The genome of the choanoflagellate Monosiga brevicollis contains a surprisingly high number and diversity of tyrosine kinases, tyrosine phosphatases, and phosphotyrosine-binding domains. Many of the tyrosine kinases possess combinations of domains that have not been observed in any multicellular organism. The role of these protein interaction domains in M. brevicollis kinase signaling is not clear. Here, we have carried out a biochemical characterization of Monosiga HMTK1, a protein containing a putative PTB domain linked to a tyrosine kinase catalytic domain. We cloned, expressed, and purified HMTK1, and we demonstrated that it possesses tyrosine kinase activity. We used immobilized peptide arrays to define a preferred ligand for the third PTB domain of HMTK1. Peptide sequences containing this ligand sequence are phosphorylated efficiently by recombinant HMTK1, suggesting that the PTB domain of HMTK1 has a role in substrate recognition analogous to the SH2 and SH3 domains of mammalian Src family kinases. We suggest that the substrate recruitment function of the noncatalytic domains of tyrosine kinases arose before their roles in autoinhibition.
    PLoS ONE 04/2011; 6(4):e19296. DOI:10.1371/journal.pone.0019296 · 3.53 Impact Factor
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    Victoria Prieto-Echagüe, W Todd Miller
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    ABSTRACT: Ack family non-receptor tyrosine kinases are unique with regard to their domain composition and regulatory properties. Human Ack1 (activated Cdc42-associated kinase) is ubiquitously expressed and is activated by signals that include growth factors and integrin-mediated cell adhesion. Stimulation leads to Ack1 autophosphorylation and to phosphorylation of additional residues in the C-terminus. The N-terminal SAM domain is required for full activation. Ack1 exerts some of its effects via protein-protein interactions that are independent of its kinase activity. In the basal state, Ack1 activity is suppressed by an intramolecular interaction between the catalytic domain and the C-terminal region. Inappropriate Ack1 activation and signaling has been implicated in the development, progression, and metastasis of several forms of cancer. Thus, there is increasing interest in Ack1 as a drug target, and studies of the regulatory properties of the enzyme may reveal features that can be exploited in inhibitor design.
    02/2011; 2011:742372. DOI:10.1155/2011/742372
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    ABSTRACT: The mechanisms that regulate the activity of the nonreceptor tyrosine kinase Ack1 (activated Cdc42-associated kinase) are poorly understood. The amino-terminal region of Ack1 is predicted to contain a sterile alpha motif (SAM) domain. SAM domains share a common fold and mediate protein-protein interactions in a wide variety of proteins. Here, we addressed the importance of the Ack1 SAM domain in kinase activity. We used immunofluorescence and Western blotting to show that Ack1 deletion mutants lacking the N-terminus displayed significantly reduced autophosphorylation in cells. A minimal construct comprising the N-terminus and kinase domain (NKD) was autophosphorylated, while the kinase domain alone (KD) was not. When expressed in mammalian cells, NKD localized to the plasma membrane, while KD showed a more diffuse cytosolic localization. Co-immunoprecipitation experiments showed a stronger interaction between full length Ack1 and NKD than between full length Ack1 and KD, indicating that the N-terminus was important for Ack1 dimerization. Increasing the local concentration of purified Ack1 kinase domain at the surface of lipid vesicles stimulated autophosphorylation and catalytic activity, consistent with a requirement for dimerization and trans-phosphorylation for activity. Collectively, the data suggest that the N-terminus of Ack1 promotes membrane localization and dimerization to allow for autophosphorylation.
    BMC Biochemistry 10/2010; 11:42. DOI:10.1186/1471-2091-11-42 · 1.94 Impact Factor
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    ABSTRACT: Ack1 is a nonreceptor tyrosine kinase that participates in tumorigenesis, cell survival, and migration. Relatively little is known about the mechanisms that regulate Ack1 activity. Recently, four somatic missense mutations of Ack1 were identified in cancer tissue samples, but the effects on Ack1 activity, and function have not been described. These mutations occur in the N-terminal region, the C-lobe of the kinase domain, and the SH3 domain. Here, we show that the cancer-associated mutations increase Ack1 autophosphorylation in mammalian cells without affecting localization and increase Ack1 activity in immune complex kinase assays. The cancer-associated mutations potentiate the ability of Ack1 to promote proliferation and migration, suggesting that point mutation is a mechanism for Ack1 deregulation. We propose that the C-terminal Mig6 homology region (MHR) (residues 802-990) participates in inhibitory intramolecular interactions. The isolated kinase domain of Ack1 interacts directly with the MHR, and the cancer-associated E346K mutation prevents binding. Likewise, mutation of a key hydrophobic residue in the MHR (Phe(820)) prevents the MHR-kinase interaction, activates Ack1, and increases cell migration. Thus, the cancer-associated mutation E346K appears to destabilize an autoinhibited conformation of Ack1, leading to constitutively high Ack1 activity.
    Journal of Biological Chemistry 04/2010; 285(14):10605-15. DOI:10.1074/jbc.M109.060459 · 4.60 Impact Factor
  • W Todd Miller
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    ABSTRACT: Biochemistry of Signal Transduction and Regulation by Gerhard Krauss is a one-volume introduction to the field of cell signaling best suited for undergraduates or beginning graduate students. The coverage of topics is broad and particularly strong regarding the regulation of gene expression in response to signaling.
    Science Signaling 12/2009; 2(101):pe79. DOI:10.1126/scisignal.2101pe79 · 7.65 Impact Factor
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    ABSTRACT: Src family kinases (SFKs) are modular signaling proteins possessing SH3, SH2, and tyrosine kinase domains. The SH3 and SH2 domains of SFKs have dual roles: they regulate the activity of the kinases, and they also target SFKs to their cellular substrates. We generated a series of novel SFKs by replacing the SH2 and SH3 domains of Hck with the syntrophin PDZ domain. In some constructs, the negative regulatory tyrosine in the C-terminal tail was also replaced with a PDZ ligand sequence. When expressed in mammalian cells, the substrate specificity of the PDZ-kinases was directed to a different group of proteins than wild-type Hck. The PDZ-kinases phosphorylate neuronal nitric oxide synthase (nNOS), a known binding partner of the syntrophin PDZ domain. We also introduced a PDZ ligand at the C-terminus of the adaptor protein Cas. PDZ-Hck kinases phosphorylate the engineered Cas protein in Cas(-/-) cells and restore the migration defect of these cells. A PDZ-kinase was also functional in rewiring MAPK signaling via an engineered ErbB2 construct containing a PDZ ligand sequence. Several of the PDZ-kinases show autoregulatory properties similar to natural SFKs. Thus, the PDZ-ligand interaction is able to functionally replace the normal SH2-pY527 interaction that regulates SFKs. Our data highlight the modularity and evolvability of signaling proteins.
    Biochemistry 10/2009; 48(46):10956-62. DOI:10.1021/bi900978f · 3.19 Impact Factor
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    ABSTRACT: The adaptor protein Cas contains a core substrate domain with multiple YXXP motifs that are phosphorylated by Src and other tyrosine kinases. Here, we used a synthetic strategy to determine the importance of the arrangement, spacing, and identity of the YXXP motifs. By polymerizing short DNA sequences encoding two phosphorylation motifs, we created a panel of Cas mutants in which the entire substrate domain was replaced by synthetic domains containing random numbers and arrangements of the motifs. Most of these synthetic Cas variants were recognized and phosphorylated by Src in vitro and in intact mammalian cells. The random polymer mutants also restored migration activity to Cas knockout cells; even artificial proteins containing a single motif retained some biological function. Our results suggest that the arrangement of Cas motifs is not critical for signaling. This method could be used to identify the minimal functional units in other signaling proteins.
    ACS Chemical Biology 09/2009; 4(9):751-8. DOI:10.1021/cb900059f · 5.36 Impact Factor

Publication Stats

3k Citations
462.44 Total Impact Points

Institutions

  • 1998–2015
    • Stony Brook University
      • • Department of Medicine
      • • Department of Physiology & Biophysics
      Stony Brook, New York, United States
  • 2013
    • Rutgers New Jersey Medical School
      Newark, New Jersey, United States
  • 2012
    • Stratford University
      Stratford, Connecticut, United States
  • 2000–2008
    • Stony Brook University Hospital
      Stony Brook, New York, United States
  • 1999–2008
    • State University of New York
      New York City, New York, United States
  • 2003
    • Albert Einstein College of Medicine
      New York, New York, United States
  • 1997–2002
    • The Rockefeller University
      • Laboratory of Molecular Biophysics
      New York City, NY, United States
  • 2001
    • University of Colorado
      Denver, Colorado, United States