Regulation of Ack-Family Nonreceptor Tyrosine Kinases

Ack family non-receptor tyrosine kinases are unique with regard to their domain composition and regulatory properties. Human Ack1 (activated Cdc42-associated kinase) is ubiquitously expressed and is activated by signals that include growth factors and integrin-mediated cell adhesion. Stimulation leads to Ack1 autophosphorylation and to phosphorylation of additional residues in the C-terminus. The N-terminal SAM domain is required for full activation. Ack1 exerts some of its effects via protein-protein interactions that are independent of its kinase activity. In the basal state, Ack1 activity is suppressed by an intramolecular interaction between the catalytic domain and the C-terminal region. Inappropriate Ack1 activation and signaling has been implicated in the development, progression, and metastasis of several forms of cancer. Thus, there is increasing interest in Ack1 as a drug target, and studies of the regulatory properties of the enzyme may reveal features that can be exploited in inhibitor design.

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Insights into the structure and protein–protein interactions of the pro-apoptotic protein ASPP2

Biochemical Society Transactions ◽

10.1042/bst0350966 ◽

2007 ◽

Vol 35 (5) ◽

pp. 966-969 ◽

Cited By ~ 10

Author(s):

S. Rotem ◽

C. Katz ◽

A. Friedler

Keyword(s):

Intermolecular Interactions ◽

Protein Interactions ◽

Intramolecular Interaction ◽

Protein Protein Interactions ◽

Sh3 Domains ◽

Src Homology 3 ◽

Rich Domain ◽

C Terminus ◽

Apoptotic Protein ◽

Src Homology

ASPP (apoptosis-stimulating protein of p53) 2 is a pro-apoptotic protein that stimulates the p53-mediated apoptotic response. Here, we provide an overview of the structure and protein–protein interactions of ASPP2. The C-terminus of ASPP2 contains Ank (ankyrin) repeats and an SH3 domain (Src homology 3 domain). The Ank–SH3 domains mediate interactions between ASPP2 and numerous proteins involved in apoptosis such as p53 and Bcl-2. The proline-rich domain of ASPP2 is unfolded in its native state, but was not shown to mediate intermolecular interactions. Instead, it makes an intramolecular domain–domain interaction with the Ank–SH3 C-terminal domains of ASPP2. This intramolecular interaction between the unstructured proline-rich domain and the structured Ank–SH3 domains in ASPP2, which is possible due to the unfolded nature of the proline-rich domain, is proposed to have an important role in regulating the intermolecular interactions of ASPP2 with its partner proteins.

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E. coli primase and DNA polymerase III holoenzyme are able to bind concurrently to a primed template during DNA replication

Scientific Reports ◽

10.1038/s41598-019-51031-0 ◽

2019 ◽

Vol 9 (1) ◽

Author(s):

Andrea Bogutzki ◽

Natalie Naue ◽

Lidia Litz ◽

Andreas Pich ◽

Ute Curth

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

Protein Interactions ◽

Dna Polymerase Iii ◽

Protein Protein Interactions ◽

Single Stranded Dna ◽

E Coli ◽

Polymerase Iii ◽

C Terminus ◽

Pol Iii

Abstract During DNA replication in E. coli, a switch between DnaG primase and DNA polymerase III holoenzyme (pol III) activities has to occur every time when the synthesis of a new Okazaki fragment starts. As both primase and the χ subunit of pol III interact with the highly conserved C-terminus of single-stranded DNA-binding protein (SSB), it had been proposed that the binding of both proteins to SSB is mutually exclusive. Using a replication system containing the origin of replication of the single-stranded DNA phage G4 (G4ori) saturated with SSB, we tested whether DnaG and pol III can bind concurrently to the primed template. We found that the addition of pol III does not lead to a displacement of primase, but to the formation of higher complexes. Even pol III-mediated primer elongation by one or several DNA nucleotides does not result in the dissociation of DnaG. About 10 nucleotides have to be added in order to displace one of the two primase molecules bound to SSB-saturated G4ori. The concurrent binding of primase and pol III is highly plausible, since even the SSB tetramer situated directly next to the 3′-terminus of the primer provides four C-termini for protein-protein interactions.

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ArabidopsisImmunophilin-like TWD1 Functionally Interacts with Vacuolar ABC Transporters

Molecular Biology of the Cell ◽

10.1091/mbc.e03-11-0831 ◽

2004 ◽

Vol 15 (7) ◽

pp. 3393-3405 ◽

Cited By ~ 70

Author(s):

Markus Geisler ◽

Marjolaine Girin ◽

Sabine Brandt ◽

Vincent Vincenzetti ◽

Sonia Plaza ◽

...

Keyword(s):

Protein Interactions ◽

Abc Transporters ◽

Loss Of Function ◽

Protein Protein Interactions ◽

Binding Motifs ◽

Calmodulin Binding ◽

C Terminus ◽

Domain Mapping ◽

Tetratricopeptide Repeat Domain

Previously, the immunophilin-like protein TWD1 from Arabidopsis has been demonstrated to interact with the ABC transporters AtPGP1 and its closest homologue, AtPGP19. Physiological and biochemical investigation of pgp1/pgp19 and of twd1 plants suggested a regulatory role of TWD1 on AtPGP1/AtPGP19 transport activities. To further understand the dramatic pleiotropic phenotype that is caused by loss-of-function mutation of the TWD1 gene, we were interested in other TWD1 interacting proteins. AtMRP1, a multidrug resistance-associated (MRP/ABCC)-like ABC transporter, has been isolated in a yeast two-hybrid screen. We demonstrate molecular interaction between TWD1 and ABC transporters AtMRP1 and its closest homologue, AtMRP2. Unlike AtPGP1, AtMRP1 binds to the C-terminal tetratricopeptide repeat domain of TWD1, which is well known to mediate protein-protein interactions. Domain mapping proved that TWD1 binds to a motif of AtMRP1 that resembles calmodulin-binding motifs; and calmodulin binding to the C-terminus of MRP1 was verified. By membrane fractionation and GFP-tagging, we localized AtMRP1 to the central vacuolar membrane and the TWD1-AtMRP1 complex was verified in vivo by coimmunoprecipitation. We were able to demonstrate that TWD1 binds to isolated vacuoles and has a significant impact on the uptake of metolachlor-GS and estradiol-β-glucuronide, well-known substrates of vacuolar transporters AtMRP1 and AtMRP2.

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Aspartic Acid Isomerization Characterized by High Definition Mass Spectrometry Significantly Alters the Bioactivity of a Novel Toxin from Poecilotheria

Toxins ◽

10.3390/toxins12040207 ◽

2020 ◽

Vol 12 (4) ◽

pp. 207 ◽

Cited By ~ 1

Author(s):

Stephen R. Johnson ◽

Hillary G. Rikli

Keyword(s):

Mass Spectrometry ◽

Protein Interactions ◽

Ion Mobility ◽

Protein Protein Interactions ◽

High Definition ◽

Vast Number ◽

Post Translational Modifications ◽

C Terminus ◽

Acid Isomerization

Research in toxinology has created a pharmacological paradox. With an estimated 220,000 venomous animals worldwide, the study of peptidyl toxins provides a vast number of effector molecules. However, due to the complexity of the protein-protein interactions, there are fewer than ten venom-derived molecules on the market. Structural characterization and identification of post-translational modifications are essential to develop biological lead structures into pharmaceuticals. Utilizing advancements in mass spectrometry, we have created a high definition approach that fuses conventional high-resolution MS-MS with ion mobility spectrometry (HDMSE) to elucidate these primary structure characteristics. We investigated venom from ten species of “tiger” spider (Genus: Poecilotheria) and discovered they contain isobaric conformers originating from non-enzymatic Asp isomerization. One conformer pair conserved in five of ten species examined, denominated PcaTX-1a and PcaTX-1b, was found to be a 36-residue peptide with a cysteine knot, an amidated C-terminus, and isoAsp33Asp substitution. Although the isomerization of Asp has been implicated in many pathologies, this is the first characterization of Asp isomerization in a toxin and demonstrates the isomerized product’s diminished physiological effects. This study establishes the value of a HDMSE approach to toxin screening and characterization.

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Divisome under Construction: Distinct Domains of the Small Membrane Protein FtsB Are Necessary for Interaction with Multiple Cell Division Proteins

Journal of Bacteriology ◽

10.1128/jb.01597-08 ◽

2009 ◽

Vol 191 (8) ◽

pp. 2815-2825 ◽

Cited By ~ 40

Author(s):

Mark D. Gonzalez ◽

Jon Beckwith

Keyword(s):

Cell Division ◽

Membrane Proteins ◽

Protein Interactions ◽

Cell Envelope ◽

Coiled Coil ◽

Main Function ◽

Protein Protein Interactions ◽

Molecular Scaffold ◽

C Terminus ◽

Under Construction

ABSTRACT Cell division in bacteria requires the coordinated action of a set of proteins, the divisome, for proper constriction of the cell envelope. Multiple protein-protein interactions are required for assembly of a stable divisome. Within the Escherichia coli divisome is a conserved subcomplex of inner membrane proteins, the FtsB/FtsL/FtsQ complex, which is necessary for linking the upstream division proteins, which are predominantly cytoplasmic, with the downstream division proteins, which are predominantly periplasmic. FtsB and FtsL are small bitopic membrane proteins with predicted coiled-coil motifs, which themselves form a stable subcomplex that can recruit downstream division proteins independently of FtsQ; however, the details of how FtsB and FtsL interact together and with other proteins remain to be characterized. Despite the small size of FtsB, we identified separate interaction domains of FtsB that are required for interaction with FtsL and FtsQ. The N-terminal half of FtsB is necessary for interaction with FtsL and sufficient, when in complex with FtsL, for recruitment of downstream division proteins, while a portion of the FtsB C terminus is necessary for interaction with FtsQ. These properties of FtsB support the proposal that its main function is as part of a molecular scaffold to allow for proper formation of the divisome.

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Insulin signalling: putting the G- in protein-protein interactions

Biochemical Journal ◽

10.1042/bj20040619 ◽

2004 ◽

Vol 380 (1) ◽

pp. e11-e12 ◽

Cited By ~ 10

Author(s):

Craig C. MALBON

Keyword(s):

Insulin Receptor ◽

Cross Talk ◽

Protein Interactions ◽

Tyrosine Kinases ◽

Cell Biology ◽

Receptor Tyrosine Kinases ◽

Insulin Signalling ◽

Cell Signalling ◽

Protein Protein Interactions ◽

Proximal Point

Cell signalling via receptor tyrosine kinases, such as the insulin receptor, and via heterotrimeric G-proteins, such as Gαi, Gαs and Gαq family members, constitute two of most avidly studied paradigms in cell biology. That elements of these two populous signalling pathways must cross-talk to achieve proper signalling in the regulation of cell proliferation, differentiation and metabolism has been anticipated, but the evolution of our thinking and the analysis of such cross-talk have lagged behind the ever-expanding troupe of players and the recognition of multivalency as the rule, rather than the exception, in signalling biology. New insights have been provided by Kreuzer et al. in this issue of the Biochemical Journal, in which insulin is shown to provoke recruitment of Gαi-proteins to insulin-receptor-based complexes that can regulate the gain of insulin-receptor-catalysed autophosphorylation, a proximal point in the insulin-sensitive cascade of signalling. Understanding the convergence and cross-talk of signals from the receptor tyrosine kinases and G-protein-coupled receptor pathways in physical, spatial and temporal contexts will remain a major challenge of cell biology.

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An activity-based probe reveals dynamic protein–protein interactions mediating IGF-1R transactivation by the GABAB receptor

Biochemical Journal ◽

10.1042/bj20120188 ◽

2012 ◽

Vol 443 (3) ◽

pp. 627-634 ◽

Cited By ~ 17

Author(s):

Xin Lin ◽

Xin Li ◽

Ming Jiang ◽

Linhai Chen ◽

Chanjuan Xu ◽

...

Keyword(s):

Protein Interactions ◽

Protein Complex ◽

Tyrosine Kinases ◽

Chemical Biology ◽

Molecular Mechanisms ◽

Gabab Receptor ◽

Type I ◽

Protein Protein Interactions ◽

Downstream Effectors ◽

Factor Type

Many GPCRs (G-protein-coupled receptors) can activate RTKs (receptor tyrosine kinases) in the absence of RTK ligands, a phenomenon called transactivation. However, the underlying molecular mechanisms remain undefined. In the present study we investigate the molecular basis of GABAB (γ-aminobutyric acid B) receptor-mediated transactivation of IGF-1R (insulin-like growth factor type I receptor) in primary neurons. We take a chemical biology approach by developing an activity-based probe targeting the GABAB receptor. This probe enables us first to lock the GABAB receptor in an inactive state and then activate it with a positive allosteric modulator, thereby permitting monitoring of the dynamic of the protein complex associated with IGF-1R transactivation. We find that activation of the GABAB receptor induces a dynamic assembly and disassembly of a protein complex, including both receptors and their downstream effectors. FAK (focal adhesion kinase), a non-RTK, plays a key role in co-ordinating this dynamic process. Importantly, this dynamic of the GABAB receptor-associated complex is critical for transactivation and transactivation-dependent neuronal survival. The present study has identified an important mechanism underlying GPCR transactivation of RTKs, which was enabled by a new chemical biology tool generally applicable for dissecting GPCR signalling.

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Proteins with SH2 and SH3 domains couple receptor tyrosine kinases to intracellular signalling pathways

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.1993.0069 ◽

1993 ◽

Vol 340 (1293) ◽

pp. 279-285 ◽

Cited By ~ 35

Keyword(s):

Protein Interactions ◽

Tyrosine Kinases ◽

Receptor Protein ◽

Signalling Pathways ◽

Protein Protein Interactions ◽

Sh3 Domains ◽

Ras Pathway ◽

Sh2 Domains ◽

Intracellular Signalling Pathways ◽

Src Homology

The targets of receptor protein-tyrosine kinases are characterized by Src homology 2 (SH2) domains, that mediate specific interactions with receptor autophosphorylation sites. SH 2-mediated interactions are important for the activation of biochemical signalling pathways in cells stimulated with growth factors. A distinct protein module, the SH3 domain, is frequently found in polypeptides that contain SH2 domains, and is also implicated in controlling protein-protein interactions in signal transduction. Evidence suggesting that SH2 and SH3 domains act synergistically in stimulation of the Ras pathway is discussed.

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Positive and Negative Regulation of Poly(A) Nuclease

Molecular and Cellular Biology ◽

10.1128/mcb.24.12.5521-5533.2004 ◽

2004 ◽

Vol 24 (12) ◽

pp. 5521-5533 ◽

Cited By ~ 57

Author(s):

David A. Mangus ◽

Matthew C. Evans ◽

Nathan S. Agrin ◽

Mandy Smith ◽

Preetam Gongidi ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Protein Interactions ◽

Binding Pocket ◽

Negative Regulation ◽

Single Amino Acid ◽

Carboxy Terminus ◽

Protein Protein Interactions ◽

C Terminus

ABSTRACT PAN, a yeast poly(A) nuclease, plays an important nuclear role in the posttranscriptional maturation of mRNA poly(A) tails. The activity of this enzyme is dependent on its Pan2p and Pan3p subunits, as well as the presence of poly(A)-binding protein (Pab1p). We have identified and characterized the associated network of factors controlling the maturation of mRNA poly(A) tails in yeast and defined its relevant protein-protein interactions. Pan3p, a positive regulator of PAN activity, interacts with Pab1p, thus providing substrate specificity for this nuclease. Pab1p also regulates poly(A) tail trimming by interacting with Pbp1p, a factor that appears to negatively regulate PAN. Pan3p and Pbp1p both interact with themselves and with the C terminus of Pab1p. However, the domains required for Pan3p and Pbp1p binding on Pab1p are distinct. Single amino acid changes that disrupt Pan3p interaction with Pab1p have been identified and define a binding pocket in helices 2 and 3 of Pab1p's carboxy terminus. The importance of these amino acids for Pab1p-Pan3p interaction, and poly(A) tail regulation, is underscored by experiments demonstrating that strains harboring substitutions in these residues accumulate mRNAs with long poly(A) tails in vivo.

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