Publication Date

2015-08-11

Availability

Embargoed

Embargo Period

2017-08-10

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Biochemistry and Molecular Biology (Medicine)

Date of Defense

2015-07-02

First Committee Member

Ralf Landgraf

Second Committee Member

Yanbin Zhang

Third Committee Member

Richard L. Rotundo

Fourth Committee Member

James N. Wilson

Abstract

Folding and maturation of most protein kinases require chaperone assistance. In higher eukaryotes, CDC37 is the predominant cochaperone that facilitates the transfer of kinase clients to Hsp90. Kinase recognition is thought to occur through the N-terminal domain, which has, thus far, eluded structure determination. Client processing requires phosphorylation of the N-terminal tail at Ser13 by the protein kinase, CK2 (casein kinase 2). How phosphorylation alters the molecular properties of CDC37 is not understood. We show that phosphorylation at Ser13 induces a large shift towards a more compact structure, based on ANS fluorescence, while modestly increasing the secondary structure. Moreover, this transition requires interactions of the N-terminal domain and the remainder of CDC37. The stabilizing property of the phosphorylation event can be recreated in trans by a (phospho-Ser13) peptide derived from the N-terminal tail. However, the phosphorylation-induced transition is not dependent on the transferred phosphate group but rather on the loss of serine-like properties at position 13. The complete absence of the N-terminal tail results in a reduced secondary structure and unresponsiveness to subsequent addition of peptides. The N-terminal tail may therefore serve as an intramolecular chaperone that ensures that CDC37 assumes one of two readily interconvertible states in a manner that impacts the interaction between the client binding N-domain and the MC-domains, involved in dimerization and Hsp90 binding. To study the role of CDC37 in structural proofreading with its client engaged, we adopted ERBB3 as a model kinase client. ERBB3 is one of four members of the ERBB receptor tyrosine kinase family. Distinct from the other ERBB members, ERBB3 has a highly impaired kinase domain with only 0.1% kinase activity compared to fully activated EGFR. Lys 723 is part of the kinase domain, and its mutation, K723M, largely diminishes the ATP binding ability of ERBB3. Despite its potential structural perturbation, K723M is successfully expressed in both insect and mammalian cells, representing itself as a valuable model for folded kinase with defects. In stable cell lines expressing either the WT or K723M ERBB3, K723M has similar mRNA levels and protein stability as WT, but demonstrates a significantly reduced steady state protein levels. In transiently transfected lines, the cell surface fraction of K723M is stable, whereas its total protein undergoes extensive degradation. We also presented that the K723M mutant of ERBB3 forms a tighter association with the CDC37/Hsp90 chaperone complex, compared to that of the wild type ERBB3. Studies of CFTR (Cystic fibrosis transmembrane conductance regulator) mutant △F508 suggest a correlation between tighter chaperone association and higher ER associated degradation. Indeed we observed low overall production of K723M. The phosphoserine mutants of CDC37 are deficient in recognizing the structural perturbation at the kinase domain of K723M. This work elucidates CDC37 as not only a kinase sorting module, but a structural defect discriminator in the structural proofreading. As a vital molecule in structural proofreading, CDC37 processes many kinases that are oncogenic or critical for cancer development. Cancer treatment via CDC37 targeting is being proposed in many circumstances. However, no available direct inhibitor has been disclosed yet. We demonstrated that Withaferin A (WA) interacts with CDC37 and disrupts the Hsp90/CDC37 complex with ERBB2. The exact binding affinity is not clear, but the effects on protein downregulation, signaling attenuation and proliferation are characterized. The ERBB3 receptor contributes to resistance in treatments that target ERBB2 receptor tyrosine kinase, and its levels represent an overall risk factor for unfavorable disease outcomes in breast cancer. In the absence of classic catalytic activity, it is not a target of pan ERBB kinase inhibitors. WA has established broad anti-cancer properties through several modes of action and has been shown to be effective against triple negative breast cancers. We found that ERBB2 overexpression renders cells hypersensitive to WA. While ERBB2 downregulation is one aspect of WA treatment, it is not causal for the elevated sensitivity. In addition WA also targets ERBB3, a crucial and highly synergistic activity in ERBB2 overexpression. WA diminishes ERBB3 receptor levels and its constitutive activation as well as its associated activation of AKT activation. The simultaneous targeting of ERBB2 and ERBB3 renders cells vulnerable, even in BT474, where direct ERBB2 targeting with Lapatinib is less effective. In conclusion, WA or its derivatives may therefore serve as a low toxicity addition to supplement ERBB2 targeted therapeutics, especially in cases where ERBB3 involvement is driving resistance or reduced overall sensitivity. The post folding structural conformation of a kinase is essential for CDC37 recognition. There is no easy method to differentiate folding status and activation states of kinases. We collaborated with Dr. James Wilson to develop fluorescence activation state specific (FAST) probes against ERBB family receptor tyrosine kinases. We contributed to the conceptual design based on ATP cocrystal structures of ERBB kinase domains and the evaluation of these novel molecules in cell settings. With the incorporation of an ERBB targeting pharmacophore and optical report element, the probe emission increases due to the hydrophobic environment and restricted geometry of ERBB kinase domain upon binding. This facilitates the analysis of receptor states at low occupancy without the removal of unbound probes. With these “Turn-On probes”, conformational properties of ERBB receptor tyrosine kinases can be readily assessed after sorting by CDC37.

Keywords

ERBB; CDC37; structural proofreading; ERAD; Withaferin A; small molecular probes

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