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Erbb3/HER3 Intracellular Domain Is Competent to Bind ATP and Catalyze Autophosphorylation

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Erbb3/HER3 Intracellular Domain Is Competent to Bind ATP and Catalyze Autophosphorylation ErbB3/HER3 intracellular domain is competent to bind ATP and catalyze autophosphorylation Fumin Shia, Shannon E. Telescob, Yingting Liub, Ravi Radhakrishnanb,1, and Mark A. Lemmona,2 aDepartment of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, and bDepartment of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104 Communicated by Joseph Schlessinger, Yale University School of Medicine, New Haven, CT, March 3, 2010 (received for review December 29, 2009) ErbB3/HER3 is one of four members of the human epidermal (N815). Initial biochemical studies concluded that ErbB3 is growth factor receptor (EGFR/HER) or ErbB receptor tyrosine kinase kinase-inactive (11, 12). The recombinant ErbB3 intracellular family. ErbB3 binds neuregulins via its extracellular region and domain was reported neither to bind ATP nor to become autop- signals primarily by heterodimerizing with ErbB2/HER2/Neu. A re- hosphorylated (12). Full-length ErbB3 expressed in insect cells cently appreciated role for ErbB3 in resistance of tumor cells to could be affinity labeled with an ATP analogue (11), but only EGFR/ErbB2-targeted therapeutics has made it a focus of attention. low-level autophosphorylation was seen that could not be However, efforts to inactivate ErbB3 therapeutically in parallel increased by adding the ErbB3 ligand neuregulin and so was with other ErbB receptors are challenging because its intracellular ascribed to endogenous insect cell kinases (11). Phosphorylation kinase domain is thought to be an inactive pseudokinase that lacks of ErbB3 (or chimerae containing its kinase domain) has several key conserved (and catalytically important) residues— been seen in immunoprecipitates from other cellular contexts including the catalytic base aspartate. We report here that, despite (13–15) but may reflect trans-phosphorylation by other ErbB these sequence alterations, ErbB3 retains sufficient kinase activity receptors (6, 11). The prevailing view, therefore, is that ErbB3 to robustly trans-autophosphorylate its intracellular region— contains an inactive “pseudokinase” (6, 16). although it is substantially less active than EGFR and does not phos- ErbB2 and ErbB3 can be activated only through heterodimer- phorylate exogenous peptides. The ErbB3 kinase domain binds ATP ization (6, 17), and both are tyrosine phosphorylated in neuregu- K μ with a d of approximately 1.1 M. We describe a crystal structure lin-induced heterodimers in cells that express only these two of ErbB3 kinase bound to an ATP analogue, which resembles the ErbB receptors (18). This observation presents a conundrum inactive EGFR and ErbB4 kinase domains (but with a shortened given current models for ErbB receptor activation, where one αC-helix). Whereas mutations that destabilize this configuration kinase domain in a dimer allosterically activates its neighbor activate EGFR and ErbB4 (and promote EGFR-dependent lung can- and itself becomes trans-autophosphorylated as a consequence cers), a similar mutation conversely inactivates ErbB3. Using quan- (3, 5). It is not clear how ErbB2 could be trans-phosphorylated tum mechanics/molecular mechanics simulations, we delineate a in ErbB2/ErbB3 heterodimers through this mechanism unless reaction pathway for ErbB3-catalyzed phosphoryl transfer that ErbB3 has kinase activity. does not require the conserved catalytic base and can be catalyzed There are additional reasons to suspect that ErbB3 might re- by the “inactive-like” configuration observed crystallographically. tain a low level of kinase activity. First, mutating the aspartate These findings suggest that ErbB3 kinase activity within receptor proposed to function as a catalytic base in the Csk tyrosine kinase dimers may be crucial for signaling and could represent an impor- (19) does not abolish (but does substantially reduce) kinase ac- tant therapeutic target. tivity. Second, kinase activity has recently been reported for other presumed pseudo- or unusual kinases, including the dimerization ∣ kinase inhibitor ∣ catalytic mechanism ∣ activation loop Ca2þ/calmodulin-activated Ser/Thr kinase (20), haspin (21), and Wnk (22)—although some other pseudokinases appear to eceptor tyrosine kinases (RTKs) from the EGF receptor be genuinely inactive (23). Here we show that the kinase domain R(EGFR) or ErbB/HER family play important roles in animal of ErbB3 retains phosphoryl-transferase activity, as evidenced by development and disease (1) and are the targets of several trans-autophosphorylation of the purified ErbB3 intracellular important therapeutic agents used clinically to treat cancer. domain. Kinase activity appears to be ∼1; 000-fold weaker than Each receptor contains a large extracellular ligand-binding region that seen for EGFR but may be sufficient for receptor trans-phos- (targeted by therapeutic antibodies), a single transmembrane phorylation in the context of an ErbB heterodimer. We demon- helix, and an intracellular tyrosine kinase domain (TKD) that strate that the ErbB3 kinase domain binds ATP with an affinity is flanked by juxtamembrane and C-terminal regulatory regions similar to that of known active kinases. We also describe the and is targeted by specific small-molecule kinase inhibitors (1, 2). Ligand binding to the extracellular region promotes homo- or Author contributions: F.S., S.E.T., Y.L., R.R., and M.A.L. designed research; F.S., S.E.T., Y.L., heterodimerization of ErbB receptors, leading to allosteric and R.R. performed research; F.S., R.R., and M.A.L. analyzed data; and F.S., R.R., and M.A.L. activation of their intracellular kinase domains through the for- wrote the paper. – mation of asymmetric dimers (3 5). Within an activated dimer, The authors declare no conflict of interest. trans the C-terminal regulatory tail is -autophosphorylated on Freely available online through the PNAS open access option. tyrosines and recruits downstream signaling molecules that con- Data deposition: The atomic coordinates for the ErbB3 kinase domain bound to AMP-PNP tain phosphotyrosine-binding Src homology-2 (SH2) domains. have been deposited in the Protein Data Bank (www.rcsb.org/pdb) with the identification ErbB3/HER3 is unique among the mammalian ErbB receptors number 3LMG. in being generally considered as kinase-inactive (6). When 1To whom correspondence may be addressed at: Department of Bioengineering, first cloned (7, 8), amino acid substitutions were noted at two University of Pennsylvania, 540 Skirkanich Hall, 210 South 33rd Street, Philadelphia, particular sites that are conserved in other known kinases (9). PA 19104. E-mail: [email protected]. A critical glutamate in helix αC of other kinases (indirectly 2To whom correspondence may be addressed at: Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, 809C Stellar-Chance involved in ATP binding) is replaced by a histidine in ErbB3 Laboratories, 422 Curie Boulevard, Philadelphia, PA 19104-6059. E-mail: mlemmon@ (H740). In addition, a conserved aspartate thought to function mail.med.upenn.edu. — as a catalytic base in protein kinases (10) deprotonating the This article contains supporting information online at www.pnas.org/cgi/content/full/ substrate hydroxyl group—is replaced with asparagine in ErbB3 1002753107/DCSupplemental. 7692–7697 ∣ PNAS ∣ April 27, 2010 ∣ vol. 107 ∣ no. 17 www.pnas.org/cgi/doi/10.1073/pnas.1002753107 Downloaded by guest on October 1, 2021 crystal structure of the ErbB3 kinase domain, which provides a A NTA-Ni vesicles +++ basis for understanding why kinase activity is reduced but not control vesicles ++ absent. ATP ++++ 116.2 Results and Discussion 94.4 Autophosphorylation of the Purified ErbB3 Intracellular Domain. Our α-pTyr initial studies of the human ErbB3 intracellular domain 66.2 (ErbB3-ICD665–1323) showed no evidence for autophosphoryla- tion activity, consistent with previous reports (12). However, kDa when ErbB3-ICD was clustered at the surface of vesicles by association of its N-terminal histidine tag with lipids bearing 116.2 nitrilotriacetate (NTA)-Ni headgroups, robust tyrosine autopho- 94.4 sphorylation could be seen in antiphosphotyrosine Western blots α -His5 (Fig. 1A). This clustering method was used previously to promote 66.2 assembly of histidine-tagged signaling receptor fragments on membrane surfaces (24) and was crucial for activating the EGFR kDa kinase domain in recent studies by Kuriyan and colleagues (5). Autophosphorylation of ErbB3-ICD required both the presence B ErbB3-ICD (wt) + of NTA-Ni lipid in the vesicles and ATP addition (Fig. 1A, two ErbB3-ICD (K723M) + + + 2þ 2þ rightmost lanes). Mn or Mg ions were also required ErbB3-TKD (wt) + A 2þ (Fig. S1 ), with Mn being slightly preferred. Consistent with ErbB3-TKD (K723M) + the presence of all ErbB3 phosphorylation sites in the C-terminal 116.2 tail, removing this region (leaving just the kinase domain: ErbB3-TKD665–1001) was accompanied by a loss of autophosphor- 94.4 α-pTyr ylation. 66.2 To confirm that the autophosphorylation shown in Fig. 1A arises from ErbB3-ICD activity, and not from contaminating 116.2 kinases, we analyzed autophosphorylation of a K723M-mutated 94.4 α-His variant under the same conditions. Lysine-723 in ErbB3 is equiva- 5 lent to K721 in the ATP-binding site of the EGFR-TKD, at which 66.2 mutations are known to abolish kinase activity (25). As shown in kDa B the second lane of Fig. 1 , a K723M mutation greatly diminished C ErbB3-TKD --WT K723M Y849E N815D ErbB3-ICD autophosphorylation. However, K723M-mutated ErbB3-ICD + +++ ++ trans ErbB3-ICD could be -phosphorylated by adding isolated ATP - 665–1001 + + +++ histidine-tagged ErbB3-TKD (in the presence of NTA-Ni 94.4 B α-pTyr BIOCHEMISTRY vesicles), as shown in lane 3 of Fig. 1 . Again, a K723M mutation 66.2 diminished this activity (lane 4 in Fig. 1 B and C). Interestingly, mutating Y849 in the activation loop of ErbB3-TKD to glutamate 94.4 α impaired its ability to trans-phosphorylate ErbB3-ICD to an even 66.2 -His5 C greater degree (Fig. 1 ), suggesting a possible role for Y849 in kDa ErbB3 regulation. Taken together, these data show that the ErbB3 kinase domain retains sufficient activity for robust ICD Fig.
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