Jeanine Amacher
Jeanine Stamp


Phosphorylation Control of the Ubiquitin Ligase Cbl is Conserved in Choanoflagellates

Jeanine F. Amacher, Helen T. Hobbs, Aaron C. Cantor, Lochan Shah, Marco-Jose Rivero, Sarah A. Mulchand and John Kuriyan

Protein Science 2018 2018 27(5):923-932. Epub 2018 Mar 2  doi: 10.1002/pro.3397     (local copy)

Abstract

Cbl proteins are E3 ubiquitin ligases specialized for the regulation of tyrosine kinases by ubiquitylation. Human Cbl proteins are activated by tyrosine phosphorylation, thus setting up a feedback loop whereby the activation of tyrosine kinases triggers their own degradation. Cbl proteins are targeted to their substrates by a phosphotyrosine-binding SH2 domain. Choanoflagellates, unicellular eukaryotes that are closely related to metazoans, also contain Cbl. The tyrosine kinase complement of choanoflagellates is distinct from that of metazoans, and it is unclear if choanoflagellate Cbl is regulated similarly to metazoan Cbl. Here, we performed structure-function studies on Cbl from the choanoflagellate species Salpingoeca rosetta, and found that it undergoes phosphorylation-dependent activation. We show that S. rosetta Cbl can be phosphorylated by S. rosetta Src kinase, and that it can ubiquitylate S. rosetta Src. We also compared the substrate selectivity of human and S. rosetta Cbl by measuring ubiquitylation of Src constructs in which Cbl-recruitment sites are placed in different contexts with respect to the kinase domain. Our results indicate that for both human and S. rosetta Cbl, ubiquitylation depends on proximity and accessibility, rather than being targeted toward specific lysine residues. Our results point to an ancient interplay between phosphotyrosine and ubiquitin signaling in the metazoan lineage.

Figures from the paper

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Figure 1 from paper

Figure 1 - Cbl domain architecture and mechanism.


Constructs used in this study are labeled and indicated by black bars.
A. Domain architecture of human c-Cbl protein.
B. Model of the phospho-activation mechanism of human c Cbl. Domains are shown as a cartoon representation, described in A. The kinase substrate is shown in cyan.
C. The domain architecture of Cbl homologues, S. rosetta Cbl, C. elegans Cbl, and D. melanogaster Cbl is shown along with the constructs used (black bars).

Figure 2 from paper

Figure 2 - TKB module structures.


Shown are the structures of the TKB modules of S. rosetta Cbl and human c-Cbl (PDB ID: 2CBL), as well as the TKB-RING segment of inactive human c-Cbl (PDB ID: 2Y1M), the RING domain is indicated by an arrow.

Figure 3 from paper

Figure 3- Structural similarity between S. rosetta Cbl (green) and human c-Cbl (yellow, PDB ID: 2Y1M).


A. The TKB modules of S. rosetta Cbl and human c-Cbl are shown in ribbon representation. The regions of high sequence identity are highlighted in black, and are localized almost exclusively to the 4H-SH2 interface and the SH2 domain. The inset figure is a zoomed in view of the 4H-SH2 interface, with the side chain residues that are identical in the two structures shown in stick representation (carbon atoms are black).
B. The binding pockets for the two tyrosine residues in the linker (Tyr 368 and Tyr 371 in human c-Cbl) are also highly conserved. Human c-Cbl (left) and S. rosetta Cbl (right) are shown in surface representation (grey), with the region containing the two conserved tyrosine residues as ribbons and the tyrosine side chains as sticks (carbons are colored yellow for c-Cbl and green for S. rosetta Cbl). The far right panel shows an overlay of the linker segments on the S. rosetta Cbl structure.

Figure 3 from paper

Figure 3- continued


C. Although the tyrosine residue corresponding to the phosphosite in human c-Cbl is not visualized in the S. rosetta Cbl structure, the binding pocket is highly conserved. Side chains that form this pocket are shown as sticks for both structures.

Figure 4 from paper

Figure 4 - Cbl proteins require phosphorylation for ubiquitylation activity.


A. Schematic of the in vitro ubiquitylation assay, using the Cbl TKB-RING module.
B. Coomassie-stained protein gel of an in vitro ubiquitylation assay showing the requirement for an active kinase (here, Src-KD) in human c-Cbl activation. A higher molecular weight smear, indicated by an arrow, is only seen in the presence of Src-KD (mw ~ 35 kDa; right lane) and not the Src-inactive protein (mw ~ 52 kDa; left lane). t= 60 m for Src-inactive and 30 m for Src-KD.
C. An anti-ubiquitin Western blot of a ubiquitylation assay using D. melanogaster Cbl reveals a requirement for active Src in Cbl activity. In the absence of Src-KD, there is a build-up of E2~Ub species at approximately 22 kDa, but no protein bands at higher molecular weight. This is in contrast to the right-hand sample, which contains Src-KD. t=30 m.
D. Coomassie-stained gels showing the results of ubiquitylation reactions using constructs containing the TKB-RING segments of S. rosetta Cbl, C. elegans Cbl, and D. melanogaster Cbl. Two lanes are shown for each experiment, including either Src-inactive (left gel panels) or Src-KD (right gel panels).

Figure 5 from paper

Figure 5 - Cbl linker-RING domains alone require phosphorylation for ubiquitylation activity.


Coomassie-stained gels showing the results of ubiquitylation assays using constructs without the TKB module, i.e., the linker-RING domains alone (see diagram of construct at top). Two lanes are shown for all experiments, for S. rosetta Cbl and C. elegans Cbl, they are t=0 (left lane) and 15 min (right). For D. melanogaster Cbl TKB-RING, the lanes are t=0 (left) and t=30 min (right). The bands corresponding to the Cbl proteins are marked with asterisks. Similar to Figure 4, a higher molecular weight smear, indicated by an arrow, is only seen in the presence of Src-KD (mw ~ 35 kDa; right gel panels) and not the Src-inactive protein (mw ~ 52 kDa; left gel panels).

Figure 6 from paper

Figure 6 - The chimeric Src kinase, linker constructs designed for ubiquitylation assays.


A. The chimeric Cbl substrates used in our ubiquitylation assays are depicted in cartoon representation for ZAP70-Src and Src-EGFR (above) or by domain architecture (below). The Src kinase domain (PDB ID: 1YOJ)29 is in cyan, with lysine residues highlighted as spheres by atom (carbons are green). The ZAP-70 linker (PBD ID: 4K2R)30 is also shown in cartoon representation (orange, with lysine residues highlighted by spheres (carbons are cyan) or a dotted line. The EGFR tail is shown as a dotted line.
B. The lysine residues of chimeric proteins in the Src kinase domain that are ubiquitylated by Cbl proteins are shown as spheres (carbons are grey if not ubiquitylated, and pink if the residue is ubiquitylated). The kinase domain is shown as a grey cartoon. Ubiquitylated residues are listed below the structure.

Figure 7 from paper

Figure 7 - Chimeric proteins are ubiquitylated at similar residues


The results from in vitro ubiquitylation assays, followed by mass spectrometry, for Src kinase or the Src chimeras and Cbl homologues from 4 species are shown. The key is at the bottom.


Supplemental figures from the paper

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Supplemental Figure 1 from paper

Supplementary Figure 1


A. An organism evolutionary tree for a number of organisms, both invertebrates (blue) and vertebrates (orange) is shown. The sequence identity for each protein, as compared to human c-Cbl, is in parenthesis following the species identifier. For the slime mold clade, RING domain identity is: P. pallidum (34%), A. subglobosum (23%), D. fasciculatum (34%), D. discoideum (34%), and D. pupureum (36%).
B. Sequence comparison reveals conservation of the critical tyrosine for activation in human c-Cbl in a number of organisms distantly related to Homo sapiens.
Supplemental Figure 2 from paper

Supplementary Figure 2 - Where is the RING domain in the S. rosetta Cbl structure?


For all, green is S. rosetta Cbl and yellow is human Cbl (PDB ID: 2Y1M)
A. On the left, the alignment of S. rosetta Cbl (green) with “inactive” (SH2 unbound, inactive conformation) human Cbl (PDB ID: 2Y1M, dark grey)25 shows remarkable structural similarity. On the right, the alignment of S. rosetta Cbl with “active” (SH2 bound, active conformation) human Cbl (PDB ID: 2CBL, grey).26
B. A coomassie stained protein gel of S. rosetta Cbl fractions following a size exclusion column is shown. The full-length S. rosetta Cbl is approximately 64 kDa.

Supplemental Figure 2 from paper

Supplementary Figure 2 - Continued


For all, green is S. rosetta Cbl and yellow is human Cbl (PDB ID: 2Y1M)
C. A coomassie-stained gel of S. rosetta Cbl crystals (5 washed crystals) reveals that the predominant species that crystallized is ~55 kDa, with smaller populations of full-length and ~42 kDa protein.
D. The alignment of human Cbl with S. rosetta Cbl structures reveals that the RING domain fits in the S. rosetta Cbl crystal lattice. Proteins are shown in surface representation, with symmetry mates in grey, and denoted by an apostrophe. Below is a zoomed in view of the region in the black box, confirming the lack of steric clash between S. rosetta Cbl, S. rosetta Cbl’, and their RING domains (mesh outlines), based on alignment.

Supplemental Figure 3 from paper

Supplementary Figure 3


A. The sequence alignment of the c-Cbl, D. melanogaster Cbl, C. elegans Cbl, and S. rosetta Cbl TKB modules is shown. Black bars indicate regions with high sequence identity between c-Cbl and S. rosetta Cbl, the two most distantly related organisms. These regions are colored black in Figure 3A.

Supplemental Figure 4 from paper

Supplementary Figure 4 - Y352F S. rosetta Cbl is not activated by Src; S. rosetta Src is ubiquitylated by S. rosetta Cbl.


A. An anti-ubiquitin Western blot following an ubiquitylation assay using human Cbl shows activation in the presence of active Src kinase.
B-C. The results by coomassie-staining B and Western blot C of ubquitylation assays run in parallel using WT or Y352F S. rosetta Cbl. In contrast to the WT protein, Y352F S. rosetta Cbl is unable to be activated in the presence of active Src kinase.
D. A coomassie-stained gel of an in vitro ubiquitylation assay of S. rosetta Src and S. rosetta Cbl is shown. The 30 min time point reveals ubiquitylation, as evidenced by the appearance of higher molecular weight bands, following addition of S. rosetta Cbl.

Supplemental Figure 5 from paper

Supplementary Figure 5 - Cbl ubiquitylation sites on ZAP-70, EGFR, and Src kinase domains.


Ubiquitylated lysine residues, identified by mass spectrometry and labeled below the structures, are shown as spheres by atom (carbons are grey if not ubiquitylated, or cyan for ZAP70, yellow for EGFR, and pink for Src if ubiquitylated) on the respective kinase domains. Below each kinase domain is the electrostatic potential surface map (blue=positive, red=negative). Scale is -5 to +5 KbT/ec. PDB IDs: ZAP70 (4K2R) EGFR (2GS2) Src (1YOJ).

Supplemental Figure 6 from paper

Supplementary Figure 6 - Ubiquitylated residues in the Src, ZAP-70, and EGFR kinase domains.


The aligned sequences of the kinase domains are shown, and ubiquitylated residues are indicated by a black box.

Supplemental Figure 7 from paper

Supplementary Figure 7 - Sequence conservation in the linkers of ZAP-70, EGFR, and Met receptor.


The linker sequences for 8 or 9 vertebrate species in the region containing the phosphotyrosine that binds Cbl SH2 were aligned and are shown for ZAP-70 (top), EGFR (middle), and Met receptor (bottom, C. mydas did not have a well annotated Met receptor). The SH2-docking tyrosine is highlighted with an arrow and similar colors indicate conservation. Tyrosine residue numbers are: ZAP-70 - human (292), mouse (290), cow (290), chicken (284), turtle (288), lizard (301), frog (284), zebrafish (276), shark (285); EGFR (with signal sequence) - human (1045, with signal sequence: 1069), mouse (1069), cow (1067), chicken (1077), turtle (1020), lizard (1076), frog (1067), zebrafish (1064), shark (1054); Met - human (1003), mouse (1001), cow (1004), chicken (1004), lizard (1005), frog (997), zebrafish (1002), shark (1045).

Supplemental Figure 8 from paper

Supplementary Figure 8 - Sequence alignment of RING domains from cIAP2 (H. sapiens) and D. discoideum Cbl or human c-Cbl and D. discoideum Cbl.


The sequences of the RING domains from human cIAP2 and D. discoideum are shown, with a sequence identity of 50%. Although the residues important for RING dimerization are only moderately conserved (indicated by asterisks, also includes Val 543 and Leu 547, which are not present in this alignment), those for the interaction with E2 (e.g., UbcH5) are highly conserved, as indicated by arrows. The critical residues for cIAP2:UbcH5 binding (Val 559, Asp 562, Leu 585, Ile 590) are absolutely conserved, as well as 1 of 2 important residues (Lys 558 and Pro 589). In contrast, the sequence alignment of the RING domains from human c-Cbl and D. discoideum Cbl are much less similar, with a sequence identity of 34%.