Cell regulation by phosphotyrosine-targeted ubiquitin ligases

Three classes of E3 ubiquitin ligases, members of the Cbl, Hakai, and SOCS-Cul5-RING ligase families, stimulate the ubiquitination of phosphotyrosine-containing proteins, including receptor and nonreceptor tyrosine kinases and their phosphorylated substrates. Because ubiquitination frequently routes proteins for degradation by the lysosome or proteasome, these E3 ligases are able to potently inhibit tyrosine kinase signaling. Their loss or mutational inactivation can contribute to cancer, autoimmunity, or endocrine disorders, such as diabetes. However, these ligases also have biological functions that are independent of their ubiquitination activity. Here we review relevant literature and then focus on more-recent developments in understanding the structures, substrates, and pathways through which the phosphotyrosine-specific ubiquitin ligases regulate diverse aspects of cell biology

SnapShot: Lysine Methylation beyond Histones

Lysine methylation is a prevalent post-translational modification (PTM) used by the cell to reversibly regulate protein function. Although it has been extensively studied in the context of histones and the associated chromatin, the remaining methyllysine proteome remains largely unexplored. This SnapShot provides an overview of the current state of lysine methylation research and its emergence as a dynamic PTM occurring on histone and non-histone proteins. Lysine methylation is a prevalent post-translational modification (PTM) used by the cell to reversibly regulate protein function. Although it has been extensively studied in the context of histones and the associated chromatin, the remaining methyllysine proteome remains largely unexplored. This SnapShot provides an overview of the current state of lysine methylation research and its emergence as a dynamic PTM occurring on histone and non-histone proteins

Phosphotyrosine recognition domains: The typical, the atypical and the versatile

SH2 domains are long known prominent players in the field of phosphotyrosine recognition within signaling protein networks. However, over the years they have been joined by an increasing number of other protein domain families that can, at least with some of their members, also recognise pTyr residues in a sequence-specific context. This superfamily of pTyr recognition modules, which includes substantial fractions of the PTB domains, as well as much smaller, or even single member fractions like the HYB domain, the PKC and PKC C2 domains and RKIP, represents a fascinating, medically relevant and hence intensely studied part of the cellular signaling architecture of metazoans. Protein tyrosine phosphorylation clearly serves a plethora of functions and pTyr recognition domains are used in a similarly wide range of interaction modes, which encompass, for example, partner protein switching, tandem recognition functionalities and the interaction with catalytically active protein domains. If looked upon closely enough, virtually no pTyr recognition and regulation event is an exact mirror image of another one in the same cell. Thus, the more we learn about the biology and ultrastructural details of pTyr recognition domains, the more does it become apparent that nature cleverly combines and varies a few basic principles to generate a sheer endless number of sophisticated and highly effective recognition/regulation events that are, under normal conditions, elegantly orchestrated in time and space. This knowledge is also valuable when exploring pTyr reader domains as diagnostic tools, drug targets or therapeutic reagents to combat human diseases. © 2012 Kaneko et al.; licensee BioMed Central Ltd

Phosphorylation-dependent inhibition of Akt1

Protein kinase B (Akt1) is a proto-oncogene that is overactive in most cancers. Akt1 activation requires phosphorylation at Thr308; phosphorylation at Ser473 further enhances catalytic activity. Akt1 activity is also regulated via interactions between the kinase domain and the N-terminal auto-inhibitory pleckstrin homology (PH) domain. As it was previously difficult to produce Akt1 in site-specific phosphorylated forms, the contribution of each activating phosphorylation site to auto-inhibition was unknown. Using a combination of genetic code expansion and in vivo enzymatic phosphorylation, we produced Akt1 variants containing programmed phosphorylation to probe the interplay between Akt1 phosphorylation status and the auto-inhibitory function of the PH domain. Deletion of the PH domain increased the enzyme activity for all three phosphorylated Akt1 variants. For the doubly phosphorylated enzyme, deletion of the PH domain relieved auto-inhibition by 295-fold. We next found that phosphorylation at Ser473 provided resistance to chemical inhibition by Akti-1/2 inhibitor VIII. The Akti-1/2 inhibitor was most effective against pAkt1T308 and showed four-fold decreased potency with Akt1 variants phosphorylated at Ser473. The data highlight the need to design more potent Akt1 inhibitors that are effective against the doubly phosphorylated and most pathogenic form of Akt1

Hdac8 activates akt through upregulating plcb1 and suppressing desc1 expression in mek1/2 inhibition-resistant cells

Inhibition of the RAF-MEK1/2-ERK signaling pathway is an ideal strategy for treating cancers with NRAS or BRAF mutations. However, the development of resistance due to incomplete inhibition of the pathway and activation of compensatory cell proliferation pathways is a major impediment of the targeted therapy. The anthrax lethal toxin (LT), which cleaves and inactivates MEKs, is a modifiable biomolecule that can be delivered selectively to tumor cells and potently kills various tumor cells. However, resistance to LT and the mechanism involved are yet to be explored. Here, we show that LT, through inhibiting MEK1/2-ERK activation, inhibits the proliferation of cancer cells with NRAS/BRAF mutations. Among them, the human colorectal tumor HT-29 and murine melanoma B16-BL6 cells developed resistance to LT in 2 to 3 days of treatment. These resistant cells activated AKT through a histone deacetylase (HDAC) 8-dependent pathway. Using an Affymetrix microarray, followed by qPCR validation, we identified that the differential expression of the phos-pholipase C-β1 (PLCB1) and squamous cell carcinoma-1 (DESC1) played an important role in HDAC8-mediated AKT activation and resistance to MEK1/2-ERK inhibition. By using inhibitors, small interference RNAs and/or expression vectors, we found that the inhibition of HDAC8 suppressed PLCB1 expression and induced DESC1 expression in the resistant cells, which led to the inhibition of AKT and re-sensitization to LT and MEK1/2 inhibition. These results suggest that targeting PLCB1 and DESC1 is a novel strategy for inhibiting the resistance to MEK1/2 inhibition

A phosphorylation switch controls the spatiotemporal activation of Rho GTPases in directional cell migration

Although cell migration plays a central role in development and disease, the underlying molecular mechanism is not fully understood. Here we report that a phosphorylationmediated molecular switch comprising deleted in liver cancer 1(DLC1), tensin-3(TNS3), phosphatase and tensin homologue (PTEN) and phosphoinositide-3-kinase (PI3K) controls the spatiotemporal activation of the small GTPases, Rac1 and RhoA, thereby initiating directional cell migration induced by growth factors. On epidermal growth factor (EGF) or platelet-derived growth factor (PDGF) stimulation, TNS3 and PTEN are phosphorylated at specific Thr residues, which trigger the rearrangement of the TNS3-DLC1 and PTEN-PI3K complexes into the TNS3-PI3K and PTEN-DLC1 complexes. Subsequently, the TNS3-PI3K complex translocates to the leading edge of a migrating cell to promote Rac1 activation, whereas PTEN-DLC1 translocates to the posterior for localized RhoA activation. Our work identifies a core signalling mechanism by which an external motility stimulus is coupled to the spatiotemporal activation of Rac1 and RhoA to drive directional cell migration

Erratum: A phosphorylation switch controls the spatiotemporal activation of Rho GTPases in directional cell migration

Although cell migration plays a central role in development and disease, the underlying molecular mechanism is not fully understood. Here we report that a phosphorylation-mediated molecular switch comprising deleted in liver cancer 1 (DLC1), tensin-3 (TNS3), phosphatase and tensin homologue (PTEN) and phosphoinositide-3-kinase (PI3K) controls the spatiotemporal activation of the small GTPases, Rac1 and RhoA, thereby initiating directional cell migration induced by growth factors. On epidermal growth factor (EGF) or platelet-derived growth factor (PDGF) stimulation, TNS3 and PTEN are phosphorylated at specific Thr residues, which trigger the rearrangement of the TNS3–DLC1 and PTEN–PI3K complexes into the TNS3–PI3K and PTEN–DLC1 complexes. Subsequently, the TNS3–PI3K complex translocates to the leading edge of a migrating cell to promote Rac1 activation, whereas PTEN–DLC1 translocates to the posterior for localized RhoA activation. Our work identifies a core signalling mechanism by which an external motility stimulus is coupled to the spatiotemporal activation of Rac1 and RhoA to drive directional cell migration

DLC1 SAM domain-binding peptides inhibit cancer cell growth and migration by inactivating RhoA

Deleted-in-liver cancer 1 (DLC1) exerts its tumor suppressive function mainly through the Rho-GTPase–activating protein (RhoGAP) domain. When activated, the domain promotes the hydrolysis of RhoA-GTP, leading to reduced cell migration. DLC1 is kept in an inactive state by an intramolecular interaction between its RhoGAP domain and the DLC1 sterile α motif (SAM) domain. We have shown previously that this autoinhibited state of DLC1 may be alleviated by tensin-3 (TNS3) or PTEN. We show here that the TNS3/PTEN-DLC1 interactions are mediated by the C2 domains of the former and the SAM domain of the latter. Intriguingly, the DLC1 SAM domain was capable of binding to specific peptide motifs within the C2 domains. Indeed, peptides containing the binding motifs were highly effective in blocking the C2-SAM domain-domain interaction. Importantly, when fused to the tat protein-transduction sequence and subsequently introduced into cells, the C2 peptides potently promoted the RhoGAP function in DLC1, leading to decreased RhoA activation and reduced tumor cell growth in soft agar and migration in response to growth factor stimulation. To facilitate the development of the C2 peptides as potential therapeutic agents, we created a cyclic version of the TNS3 C2 domain-derived peptide and showed that this peptide readily entered the MDA-MB-231 breast cancer cells and effectively inhibited their migration. Our work shows, for the first time, that the SAM domain is a peptide-binding module and establishes the framework on which to explore DLC1 SAM domain-binding peptides as potential therapeutic agents for cancer treatment

MMS2plot: An R Package for Visualizing Multiple MS/MS Spectra for Groups of Modified and Non-Modified Peptides

A large number of post-translational modifications (PTMs) in proteins are buried in the unassigned mass spectrometric (MS) spectra in shot-gun proteomics datasets. Because the modified peptide fragments are low in abundance relative to the corresponding non-modified versions, it is critical to develop tools that allow facile evaluation of assignment of PTMs based on the MS/MS spectra. Such tools will preferably have the ability to allow comparison of fragment ion spectra and retention time between the modified and unmodified peptide pairs or group. Herein, MMS2plot, an R package for visualizing peptide-spectrum matches (PSMs) for multiple peptides, is described. MMS2plot features a batch mode and generates the output images in vector graphics file format that facilitate evaluation and publication of the PSM assignment. MMS2plot is expected to play an important role in PTM discovery from large-scale proteomics datasets generated by liquid chromatography-MS/MS. The MMS2plot package is freely available at https://github.com/lileir/MMS2plot under the GPL-3 license

Dynamic interplay of two molecular switches enabled by the MEK1/2–ERK1/2 and IL-6–STAT3 signaling axes controls epithelial cell migration in response to growth factors

Cell migration is an essential physiological process, and aberrant migration of epithelial cells underlies many pathological conditions. However, the molecular mechanisms governing cell migration are not fully understood. We report here that growth factor–induced epithelial cell migration is critically dependent on the crosstalk of two molecular switches, namely phosphorylation switch (P-switch) and transcriptional switch (T-switch). P-switch refers to dynamic interactions of deleted in liver cancer 1 (DLC1) and PI3K with tensin-3 (TNS3), phosphatase and tensin homolog (PTEN), C-terminal tension, and vav guanine nucleotide exchange factor 2 (VAV2) that are dictated by mitogen-activated protein kinase kinase 1/2–extracellular signal–regulated protein kinase 1/2–dependent phosphorylation of TNS3, PTEN, and VAV2. Phosphorylation of TNS3 and PTEN on specific Thr residues led to the switch of DLC1–TNS3 and PI3K–PTEN complexes to DLC1–PTEN and PI3K–TNS3 complexes, whereas Ser phosphorylation of VAV2 promotes the transition of the PI3K–TNS3/PTEN complexes to PI3K–VAV2 complex. T-switch denotes an increase in C-terminal tension transcription/ expression regulated by both extracellular signal–regulated protein kinase 1/2 and signal transducer and activator of transcription 3 (STAT3) via interleukin-6–Janus kinase–STAT3 signaling pathway. We have found that, the P-switch is indispensable for both the initiation and continuation of cell migration induced by growth factors, whereas the T-switch is only required to sustain cell migration. The interplay of the two switches facilitated by the interleukin-6–Janus kinase–STAT3 pathway governs a sequence of dynamic protein–protein interactions for sustained cell migration. That a similar mechanism is employed by both normal and tumorigenic epithelial cells to drive their respective migration suggests that the P-switch and T-switch are general regulators of epithelial cell migration and potential therapeutic targets