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Jinhua Wu, PhD

Jinhua Wu, PhD
About

Associate Professor

Research Program

Lab Overview

 Integrin activation mediated by intracellular signaling proteins. Activated Rap1 (orange oval) recruits RIAM via the RA-PH (blue oval) to the PM. The TBS segment (blue helix) of RIAM interacts with the rod region (orange cylinders with yellow oval background) of talin, allowing talin head domain (subdomains F0 in red, F1 in orange, F2 in green, and F3 in yellow, with yellow oval background) to bind to integrin <β> tail. Together with kindlin (grey oval), talin induces integrin activation and clustering on the extracellular matrix (ECM). RIAM:Rap1 structure is adapted from PDB code 4KVG. TBS:talin-rod structure is adapted from PDB code 4W8P. FTH:integrin-<β> is adapted from PDB code 6VGU. Integrin activation mediated by intracellular signaling proteins. Activated Rap1 (orange oval) recruits RIAM via the RA-PH (blue oval) to the PM. The TBS segment (blue helix) of RIAM interacts with the rod region (orange cylinders with yellow oval background) of talin, allowing talin head domain (subdomains F0 in red, F1 in orange, F2 in green, and F3 in yellow, with yellow oval background) to bind to integrin <β> tail. Together with kindlin (grey oval), talin induces integrin activation and clustering on the extracellular matrix (ECM). RIAM:Rap1 structure is adapted from PDB code 4KVG. TBS:talin-rod structure is adapted from PDB code 4W8P. FTH:integrin-<β> is adapted from PDB code 6VGU.

My laboratory is interested in the molecular mechanisms underlying cell adhesion and motility mediated by integrin signaling and small GTPase signaling. Integrins are cell adhesion molecules that transduce signals across the plasma membrane (PM)  bi-directionally. Misregulation of integrin expression and its activity may result in severe diseases such as autoimmune diseases, thrombotic disorders, cardiovascular diseases (CVDs), and  T cell proliferation defects. The bi-directional signaling property of integrins makes both extracellular and intracellular intervention possible for clinical benefits. Currently, the common integrin-intervention strategy is to antagonize extracellular ligand binding. These antagonistic agents have shown effects in treating thrombosis and multiple sclerosis. Nevertheless, due to the lack of specificity in recognizing integrin species and other unknown properties, serious adverse effects have been reported to associate with use of integrin antagonists. Alternatively, integrin activities can be well regulated by intracellular signaling proteins in a species-specific manner. Thus, targeting these intracellular proteins, alone or in combination with the antagonistic intervention, has the potential to achieve greater therapeutic outcomes by better differentiating integrin species and preventing unwanted signaling events. 

 

Fig. 1
Figure 1. Molecular Mechanisms of the Inside-out Integrin Signaling Pathway
Fig. 2
Figure 2. Molecular mechanisms governing activation of integrin by MRL proteins
Fig. 3
Figure 3. Interaction of TBS1 with talin rod region. A. Crystal structure of talin R7R8 in complex with RIAM TBS1 peptide. TBS1-interacting helices in R8 are 2 and 3. Hydrogen bond network mediated Ser13 is shown in the close-in view. B. Hydrophobic interactions are represented by a light gray surface with residues of TBS1 in blue stroke and residues of R8 in yellow stroke. C. Hydrogen bonds are denoted by a dotted line.
Fig. 4
Figure 4. (Left) CHO cells stably expression integrin alphaIIb/beta3 were transiently transfected with mCherry-talin and GFP-RIAM-TBS1-CAAX and its mutants. The cells were plated on fibrinogen-coated coverslips and allowed to adhere and spread. Yellow colors in merged images represent areas of common red and green localizations. (Right) Enlarged lamellae sections correspond to the white rectangles marked in the left. Note that small histogram graphs are representative of intensity levels of the assorted overexpressed proteins. While WT RIAM-TBS1 and talin colocalized at the lamellae, the mutants failed to recruit talin into the lamellae.
Fig. 5
Figure 5. Interaction of TBS1 with talin head region. A. NMR structure of talin F3 domain in complex with the RIAM TBS1 peptide. The side chains of the residues involved in the interaction are labeled. B. The structure of talin-F3 (blue) bound to RIAM-TBS1 (salmon) was superimposed onto the talin-F3 (green) bound to talin-R9 (gray). TBS1 sterically interferes with the autoinhibitory interface of F3:R9. C. Superposition of TBS1 bound talin-F3 with integrin- (yellow) bound talin-F3 indicates that TBS1 binds to a neighboring region of the integrin-binding site on talin-F3. D. Superposition of the TBS1 fragments in complex with R8 of talin-R (purple:green) or with F3 of talin-H (salmon:blue).
Postdoc Position Available

A postdoctoral position is available at Fox Chase Cancer Center (Philadelphia, PA) for a highly motivated individual in the field of protein biochemistry and cell signaling.

The main focus of the lab is to understand the molecular basis for the cell signaling events that affect integrin activity. For this, we employ a combination of techniques including binding kinetics analysis, functional assays in mammalian cells, protein crystallography, electron microscopy, and genetic analysis. Our study will contribute significantly to the understanding of the formation and regulation of cell migration and adhesion, and will facilitate efforts to discover novel targets for therapeutic intervention of tumor metastasis and other related diseases. For more details, see Research.

The candidate should have strong experiences in protein biochemistry, molecular biology, and mammalian cell culture. Prior experiences in flow cytometry, X-ray crystallography, receptor biochemistry, and cancer biology, are desired but not required. The candidate should receive a PhD degree within the last three years. Fox Chase Cancer Center is a long-standing NCI-designated Comprehensive Cancer Center with multidisciplinary research interests from basic biological sciences to drug development and clinical studies. The institute is one of the top-ranked work places for postdoctoral researchers. It offers an outstanding research environment and strong support for postdoctoral career development.

Please send a cover letter describing previous research experience and current interests, an updated CV, and a list of three references to: Jinhua.wu@fccc.edu.

Education and Training

Educational Background

  • PhD, Structural Biology, Purdue University, West Lafayette, IN, 2004
  • BS, Biology, University of Science and Technology of China, Hefei, China, 1997

Memberships

  • Member, American Society for Cell Biology 2017
  • Health Research Formula Grant Award, Pennsylvania Department of Health 2015
  • Member, American Cancer Society 2014 
  • American Cancer Society Institutional Research Grant Pilot Project Award 2012
  • Member, Fox Chase Cancer Center Cell Signaling Group 2010 
  • Member, Fox Chase Cancer Center Head and Neck Keystone 2010 
  • Member, Fox Chase Cancer Center Molecular Modeling Facility Advisory Committee 2010 
  • Member, Sigma Xi Scientific Research Society 2007 - 2008
  • Member, American Association for the Advancement of Science 2007 - 2008
  • Member, New York Structural Biology Discussion Group 2005
  • Member, American Crystallographic Association  2001 - 2007
Research Profile

Research Program

Research Interests

Structural and Functional studies of integrin signaling

  • Structural  basis to the regulatory mechanisms of the inside-out integrin signaling.
  • Structural and functional analyses of the MRL family proteins.
  • Development of anti-talin inhibitors by structure-based computer-aided design.

Lab Description

Alterations in the regulation or expression of integrins have been implicated in many human diseases including inflammatory disorders, cardiovascular diseases, and cancer. In cancer biology, altered expression of integrins has been linked to tumor cell proliferation, metastasis and survival due to their role as adhesion receptors. Hence the integrin signaling pathway has become an appealing target for cancer therapy. Integrins can be activated by extracellular ligand binding in an outside-in manner or by growth factor stimulation through an inside-out pathway. Although antagonistic inhibitors have been shown to reduce early tumor angiogenesis, the inconsistent effect remain major obstacles for anti-cancer drug development. Alternatively, Integrin activities may also be suppressed by blocking the specific interactions of intracellular elements in the inside-out signaling.

The main focus of my lab is to understand the structural basis of intermolecular complexes and intramolecular rearrangements that control integrin-mediated cell adhesion and motility. Understanding the structural details of each signaling event, particularly the protein-protein interactions involved in this pathway, is the key to developing next-generation inhibitors. We aim to elucidate the structural basis of cytosolic inter-molecular complexes and intra-molecular domain rearrangements that modify the integrin activation, to validate these specific interactions revealed in the crystal structures, and to assess their roles in integrin activation in biochemical and physiological contexts. Our lab employs a combination of X-ray crystallography, biochemical and biophysical assays, cell-based functional studies, computer modeling, and organic synthesis to accomplish these goals.

 

Project 1. Regulatory mechanisms of RIAM

RIAM autoinhibition and activation. The RA (yellow) and PH (cyan) segments of RIAM drive the PM (yellow/red surface) translocation of RIAM, which requires the interactions of Rap1(gray):RA and PI(4,5)P2:PH. The “IN” (red helix) inhibits the RAP1:RA interaction, and the PI(4,5)P2:PH interaction is suppressed by homo-oligomeric interaction, in which the PI(4,5)P2 binding site is masked by the PH segment in the RIAM oligomer (cyan surface). The two binding sites can be unmasked by FAK and Src phosphorylation, respectively. The IN-RA-PH structure is adopted from PDB code: 6E31.RIAM autoinhibition and activation. The RA (yellow) and PH (cyan) segments of RIAM drive the PM (yellow/red surface) translocation of RIAM, which requires the interactions of Rap1(gray):RA and PI(4,5)P2:PH. The “IN” (red helix) inhibits the RAP1:RA interaction, and the PI(4,5)P2:PH interaction is suppressed by homo-oligomeric interaction, in which the PI(4,5)P2 binding site is masked by the PH segment in the RIAM oligomer (cyan surface). The two binding sites can be unmasked by FAK and Src phosphorylation, respectively. The IN-RA-PH structure is adopted from PDB code: 6E31.

RAP1-interacting adapter molecule (RIAM) mediates RAP1-induced integrin activation. This function requires the translocation of RIAM to the plasma membrane (PM). The RAS-association (RA) segment of the RA-PH module of RIAM interacts with GTP-bound RAP1 and the PH segment of the RA-PH module interacts with phosphoinositol 4,5 bisphosphate (PI(4,5)P2), thus driving RIAM to the PM. The interaction of RAP1 and the RA d but this interaction is inhibited by an N-terminal segment, IN, of RIAM. We reported structural basis for the autoinhibition of RIAM by an intramolecular interaction between the IN region (aa 27-93) and the RA-PH module. We solved the crystal structure of IN-RA-RH, which reveals that the “IN” segment associates with the RA segment and thereby suppresses RIAM:RAP1 association. This autoinhibitory configuration of RIAM can be released by phosphorylation at Tyr45 in the IN segment. We then found the PH:PI(4,5)P2 interaction is also autoinhibited by a conserved inter-molecular interface that masks the PIP2 binding site in the PH domains of RIAM. Our data indicate that phosphorylation of RIAM by Src family kinases disrupts this PH-mediated interface, unmasks the membrane PIP2-binding site, and promotes integrin activation. We further demonstrate that this process requires phosphorylation of Tyr267 and Tyr427 in the RIAM PH domain by Src. Our data reveal an unorthodox regulatory mechanism of small GTPase effector proteins by phosphorylation-dependent PM association, and provide new insights into the link between FAK/Src kinases and integrin signaling.

 

Project 2. Active form of talin head

Crystal structure of talin head in a FERM-folded configuration. Overall structure of talin head domain in complex with integrin β3 peptide (PDB code: 6VGU). Talin head is shown in cartoon and surface. The F0 domain is in red. The F1, F2, and F3 subdomains of the FERM domain are in orange, green, and yellow, respectively. Bound β3 is shown in stick representation.Crystal structure of talin head in a FERM-folded configuration. Overall structure of talin head domain in complex with integrin β3 peptide (PDB code: 6VGU). Talin head is shown in cartoon and surface. The F0 domain is in red. The F1, F2, and F3 subdomains of the FERM domain are in orange, green, and yellow, respectively. Bound β3 is shown in stick representation.

Binding of the intracellular adapter proteins talin and its cofactor, kindlin, to the integrin receptors induces integrin activation and clustering. These processes are essential for cell adhesion, migration, and organ development. Although the talin head, the integrin-binding segment in talin, possesses a typical FERM-domain sequence, a truncated form has been crystallized in an unexpected, elongated form. Our crystal structure of a full-length talin head in complex with the β3-integrin tail reveals a compact FERM-like conformation and a tightly associated N-P-L-Y motif of β3-integrin. A critical C-terminal poly-lysine motif, which is truncated in the linear form, mediates FERM interdomain contacts and assures the tight association with the β3-integrin cytoplasmic segment. Therefore, structural characterization of the FERM-folded active talin head provides fundamental understanding of the regulatory mechanism of integrin function, and may lead to rediscovery of the structural basis for the talin head-mediated signal transduction.

 

Project 3. Structural and functional studies of Kindlin.

A. Crystal structure of kindlin-2 (K2ΔPH). B. F1 loop is indicated by dashed-line. C. EM classes and 3D model of kindlin-2.A. Crystal structure of kindlin-2 (K2ΔPH). B. F1 loop is indicated by dashed-line. C. EM classes and 3D model of kindlin-2.

The kindlin family includes three members, kindlin-1, kindlin-2, and kindlin-3. Kindlin possess a FERM domain similar to talin head,  and a PH domain. While the β1 and β3 integrin CTs are the common binding partners of Kindlins, each Kindlin targets a distinct integrin β tail. For instance, kindlin-3 interacts with β2 integrin. In the keratinocytes, only Kindlin-1, but not Kindlin-2, can interact with β6 integrin. Kindlin-2 recognizes β7 integrin, but not β2 or β6 integrin in vitro. We have determined the crystal structure of the complete FERM domain of kindlin-2, with the PH domain removed for crystallization purpose (K2DPH). The structure revealed a canonical FERM domain architecture, in which the F1 domain sits on top of the F2F3 domains. Compared with the published kindlin-2 structure (PDB code: 5XPY), our structure affords the full-length F1 subdomain, allowing us to obtain more structural detail of the F1 loop, which is required for kindlin-mediated integrin co-activation. Moreover, we have obtained preliminary electron microscopic data for kindlin-2. Our data reveal a monomeric model for kindlin-2. Further analyses of the EM models and the oligomerization states of kindlin isoforms are required to understand how oligomerization impacts its activity.

 

Project 4. Regulatory mechanisms of talin by intramolecular interactions

A. Crystal structure of R9R10. R9R10 structure (green) is superimposed with the structure of F2F3 (in yellow) in complex with the R9 domain (in cyan). A putative interaction between the R10 and the F3 domains is highlighted in the inset box. B. NMR structures of two R3 domains (1U89: <α>1-<α>4; 2L7A: <α>2-<α>5). C. Two crystal forms of R1R2R3.A. Crystal structure of R9R10. R9R10 structure (green) is superimposed with the structure of F2F3 (in yellow) in complex with the R9 domain (in cyan). A putative interaction between the R10 and the F3 domains is highlighted in the inset box. B. NMR structures of two R3 domains (1U89: <α>1-<α>4; 2L7A: <α>2-<α>5). C. Two crystal forms of R1R2R3.

Talin possesses a long, multi-domain, C-terminal “rod” region. Talin rod domains are helical bundle domains and regulate talin function by interacting the talin head domain and other signaling partners. Talin head interacts with the R9 domain in the rod region. To assess the possible intramolecular interactions between the rod domain adjacent to R9 and talin head, we generated a construct for the R9R10 tandem domains and determined its crystal structure. Both R9 and R10 domains are 5-helical bundles. Interestingly, the structure reveals that the last a-helix the R9 domain and the first a-helix of the R10 domain join together and form an extended helix that bridges the two domains (Figure 5A). Moreover, superposition of the R9R10 tandem domain and the R9:F2F3 autoinhibitory complex reveals potential interactions between the R10 and F3 domain (Figure 5A). In particular, Asp1943 of R10 is in close proximity to Lys364 of F3 with a distance of 5.6-Å in the superimposed models. Our data suggest that the R10 domain may also contribute to the head:rod autoinhibitory interaction. Further analyses are underway to validate this model. 

In an effort to elucidate the intramolecular interaction in talin between the R1R2R3 triple domains and the F2F3 domains in the head region, we expressed and purified R1R2R3 triple domains. Previously reported NMR structures of the R3 domains revealed two different helical-bundle arrangements (PDB codes: 1U89 and 2L7A). 1U89 is composed of residues 755-889 (a1-a4), whereas 2L7A is composed of residues 788-911 (a2-a5) (Figure 2B). Although the two helical-bundles can be superimposed nicely, a1 helix in 1U89 is replaced by a5 in 2L7A. Moreover, it has been proposed that the R1R2R3 domains may form a rather compact module instead of a “beads-on-a-string” architecture. We have obtained crystals for R1R2R3 and are in the process of determining the structure of this triple domains (Figure 5C). Our result will yield essential insight to these questions.

 
Lab Alumni
  • Dr. Pingfeng Zhang – Postdoc (Professor, Wuhan University, China)
  • Dr. Hao Zhang – Postdoc (Scientist, Frontage Lab, Inc)
  • Dr. Eric (Yu-Chung) Chang – Postdoc (Manager, Genscript)
  • Tejash Patel – Graduate Student (PCOM)
  • Mark Brennan – Scientific Technician (Scientist, GSK)
  • Pavan Patel – Scientific Technician (Med School)
  • Rebecca Stronk – Scientific Technician
    (Current Position
Lab Staff

Baihao Su, PhD

Postdoctoral Associate

Room: W410
215-728-3856

Eun-Ah Cho, PhD

Postdoctoral Associate

Room: W410
215-728-3856

Tracy (Yijuan) Sun

Visiting Scientist

Room: W410
215-728-3856

Tong Gao, PhD

Postdoctoral Associate

Room: W410
215-728-3856
Publications

Selected Publications

Zhang P, Azizi L, Kukkurainen S, Gao T, Baikoghli M, Jacquier MC, Sun Y, Maatta JAE, Cheng RH, Wehrle-Haller B, Hytonen VP, Wu J. Crystal structure of the FERM-folded talin head reveals the determinants for integrin binding. Proc Natl Acad Sci U S A. 2020. Epub 2020/12/09. doi: 10.1073/pnas.2014583117. PubMed PMID: 33288722.

Kukkurainen S, Azizi L, Zhang P, Jacquier MC, Baikoghli M, von Essen M, Tuukkanen A, Laitaoja M, Liu X, Rahikainen R, Orlowski A, Janis J, Maatta JAE, Varjosalo M, Vattulainen I, Rog T, Svergun D, Cheng RH, Wu J, Hytonen VP, Wehrle-Haller B. The F1 loop of the talin head domain acts as a gatekeeper in integrin activation and clustering. J Cell Sci. 2020;133(19). Epub 2020/10/14. doi: 10.1242/jcs.239202. PubMed PMID: 33046605.

Cho EA, Zhang P, Kumar V, Kavalchuk M, Zhang H, Huang Q, Duncan JS, Wu J. Phosphorylation of RIAM by Src Promotes Integrin Activation by Unmasking the PH Domain of RIAM. Structure. 2020. Epub 2020/12/05. doi: 10.1016/j.str.2020.11.011. PubMed PMID: 33275877.

Chang YC, Su W, Cho EA, Zhang H, Huang Q, Philips MR, Wu J. Molecular basis for autoinhibition of RIAM regulated by FAK in integrin activation. Proc Natl Acad Sci U S A. 2019;116(9):3524-9. doi: 10.1073/pnas.1818880116. PubMed PMID: 30733287; PMCID: PMC6397589.

Zhang H, Chang YC, Huang Q, Brennan ML, Wu J. Structural and Functional Analysis of a Talin Triple-Domain Module Suggests an Alternative Talin Autoinhibitory Configuration Structure. 2016 24(5):721-9. PubMed

Yang J, Zhu L, Zhang H, Hirbawi J, Fukuda K, Dwivedi P, Liu J, Byzova T, Plow EF, Wu J**, Qin J**. Conformational activation of talin by RIAM triggers integrin-mediated cell adhesion. Nat Comm Epub 2014 Dec 18. PubMed

Chang YC, Zhang H, Brennan ML, Franco-Barraza J, Patel T, Cukierman E, Wu J. Structural and mechanistic insights into the recruitment of talin by RIAM in integrin signaling. Structure 2014 22(12):1810-20. PubMed

Zhang H, Chang YC, Brennan ML, Wu J. The structure of Rap1 in complex with RIAM reveals specificity determinants and recruitment mechanism. J Mol Cell Biol 2014 6(2):128-39. PubMed

Anastassiadis T, Duong-Ly KC, Deacon SW, Lafontant A, Ma H, Devarajan K, Dunbrack RL Jr, Wu J, Peterson JR. A highly selective dual insulin receptor (IR)/insulin-like growth factor 1 receptor (IGF-1R) inhibitor derived from an extracellular signal-regulated kinase (ERK) inhibitor. J Biol Chem. 2013 Sep 27;288(39):28068-77. doi: 10.1074/jbc.M113.505032. Epub 2013 Aug 9. PubMed

Chang YC, Zhang H, Brennan ML, Wu J. Crystal structure of Lamellipodin implicates diverse functions in actin polymerization and Ras signaling. Protein Cell. 2013 Mar;4(3):211-9. doi: 10.1007/s13238-013-2082-5. Epub 2013 Mar 13. PubMed

Wynne JP*, Wu J*, Su W, Mor A, Patsoukis N, Boussiotis VA, Hubbard SR, Philips MR. Rap1-interacting adapter molecule (RIAM) associates with the plasma membrane via a proximity detector. J Cell Biol. 2012 Oct 15;199(2):317-30. doi: 10.1083/jcb.201201157. Epub 2012 Oct 8. *Equal contribution. PubMed

Ungureanu D, Wu J, Pekkala T, Niranjan Y, Young C, Jensen ON, Xu CF, Neubert TA, Skoda RC, Hubbard SR, Silvennoinen O. The pseudokinase domain of JAK2 is a dual-specificity protein kinase that negatively regulates cytokine signaling. Nat Struct Mol Biol. 2011 Aug 14. [Epub ahead of print] PubMed

Depetris RS*, Wu J*, Hubbard, S.R. Structural and functional studies of the Ras-associating and pleckstrin-homology domains of Grb10 and Grb14. Nat Struct Mol Biol. 2009;16,833-9. *Equal contribution. PubMed

Wu J*, Li W*, Craddock BP, Foreman KW, Mulvihill MJ, Ji QS, Miller WT, Hubbard SR. Small-molecule inhibition and activation-loop trans-phosphorylation of the IGF1 receptor. EMBO J. 2008;27,1985-94. *Equal contribution. PubMed

Wu J, Tseng Y, Xu C, Neubert TA, White MF, and Hubbard SR. Structural and biochemical characterization of the KRLB region in insulin receptor substrate-2. Nat Struct Mol Biol. 2008;15,251-8. PubMed

 

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