Interactome Mapping Uncovers a General Role for Numb in Protein Kinase Regulation

Abstract

Cellular functions are frequently regulated by protein-protein interactions involving the binding of a modular domain in one protein to a specific peptide sequence in another. This mechanism may be explored to identify binding partners for proteins harboring a peptide-recognition domain. Here we report a proteomic strategy combining peptide and protein microarray screening with biochemical and cellular assays to identify modular domain-mediated protein-protein interactions in a systematic manner. We applied this strategy to Numb, a multi-functional protein containing a phosphotyrosine-binding (PTB) domain. Through the screening of a protein microarray, we identified >100 protein kinases, including both Tyr and Ser/Thr kinases, that could potentially interact with the Numb PTB domain, suggesting a general role for Numb in regulating kinase function. The putative interactions between Numb and several tyrosine kinases were subsequently validated by GST pull-down and/or co-immunoprecipitation assays. Furthermore, using the Oriented Peptide Array Library approach, we defined the specificity of the Numb PTB domain which, in turn, allowed us to predict binding partners for Numb at the genome level. The combination of the protein microarray screening with computer-aided prediction produced the most expansive interactome for Numb to date, implicating Numb in regulating phosphorylation signaling through protein kinases and phosphatases. Not only does the data generated from this study provide an important resource for hypothesis-driven research to further define the function of Numb, the proteomic strategy described herein may be employed to uncover the interactome for other peptide-recognition domains whose consensus motifs are known or can be determined.

Keywords: Numb; PTB domain; Peptide array; Protein array; Protein kinases; Protein-Protein Interactions; SMALI; Tyrosine Kinases.

Fig. 1.

Systematic identification of Numb-binding proteins. A workflow highlighting the strategy used in this study to identify proteins that bind specifically to the Numb PTB domain.

Fig. 2.

A Numb PTB interactome identified by screening a protein microarray. A, B, Representative sections of a protein microarray (ProtoArray, Invitrogen) probed with the dNumb PTB domain. Protein spots that showed positive binding (red dots) were labeled. Control 1 spots represent Alexa Fluor 647 Rabbit anti-mouse IgG antibody; Control 2 spots represent Biotinylated anti-mouse antibody. C, A Venn diagram illustrating all potential binders of the dNumb or hNumb PTB domains identified from screening the ProtoArray and their overlap. D–F, Venn diagrams showing the protein kinases that bound the dNumb or/and the hNumb PTB domain.

Fig. 3.

Verification of Numb-PTB interactions by peptide array and in-solution binding. A, Binding profile for the dNumb PTB domain on an array of NxxF/Y motif-containing peptides. The peptides were derived from the PTB-interacting proteins identified using the ProtoArray (Fig. 2). B, Distribution of Z scores for the positive binders on the peptide array in (A). C–K, Binding curves of selected peptides from the peptide array for the dNumb PTB domain. See Table I for peptide sequences and K_d values.

Fig. 4.

Numb or its PTB domain binds to tyrosine kinases in cells. A, ERBB2 co-immunoprecipitated with Numb in SK-BR3 cells. B–F, The Numb PTB domains pulled down ALK, ROS1, FGR, RET and FLT3, respectively, from cells expressing these proteins.

Fig. 5.

Defining Numb PTB domain specificity by OPAL. A, Binding pattern of the dNumb PTB domain on an OPAL membrane with the degenerate sequence xxNxx[F/Y]xx. B, A box plot showing the distribution of true binders (Table I) and non-binders (from Fig. 3A) relative to the corresponding SMALI score. The medium value for true binders and non-binders are shown. C–I, Binding curves for representative peptides with a SMALI score > 1.3. See Table I for peptide sequences and K_d values.

Fig. 6.

The Numb interactome is enriched for proteins involved in phosphorylation signaling. A, Molecular function enrichment for the Numb-binding proteins identified in this study. B, Distribution of Numb-binding TKs in the human kinome phylogeny tree. Fig. reproduced courtesy of Cell Signaling Technology, Inc.