Regulation of Src and Csk nonreceptor tyrosine kinases in the filasterean Ministeria vibrans.

The development of the phosphotyrosine-based signaling system predated the evolution of multicellular animals. Single-celled choanoflagellates, the closest living relatives to metazoans, possess numerous tyrosine kinases, including Src family nonreceptor tyrosine kinases. Choanoflagellates also have Csk (C-terminal Src kinase), the enzyme that regulates Src in metazoans; however, choanoflagellate Csk kinases fail to repress the cognate Src. Here, we have cloned and characterized Src and Csk kinases from Ministeria vibrans, a filasterean (the sister group to metazoans and choanoflagellates). The two Src kinases (MvSrc1 and MvSrc2) are enzymatically active Src kinases, although they have low activity toward mammalian cellular proteins. Unexpectedly, MvSrc2 has significant Ser/Thr kinase activity. The Csk homologue (MvCsk) is enzymatically inactive and fails to repress MvSrc activity. We suggest that the low activity of MvCsk is due to sequences in the SH2-kinase interface, and we show that a point mutation in this region partially restores MvCsk activity. The inactivity of filasterean Csk kinases is consistent with a model in which the stringent regulation of Src family kinases arose more recently in evolution, after the split between choanoflagellates and multicellular animals.

Effects of heterologous kinase domains on growth factor receptor specificity.

The kinase domains of receptor tyrosine kinases (RTKs) are highly conserved, yet they are able to discriminate among potential substrates to selectively activate downstream signaling pathways. In this study, we tested the importance of catalytic domain specificity by creating two series of chimeric RTKs. In one set, the kinase domain of insulin-like growth factor I receptor (IGF1R) was replaced by the kinase domains from insulin receptor (IR), macrophage stimulating protein 1 receptor/Ron (Ron) or Src. In the other set of chimeras, the kinase domain of epidermal growth factor receptor (EGFR) was similarly replaced by the kinase domains of IR, Ron, or Src. We expressed the wild-type and chimeric forms of the receptors in mammalian cells. For some signaling events, such as recognition of IRS1, the identity of the tyrosine kinase catalytic domain did not appear to be crucial. In contrast, recognition of some sites, such as the C-terminal autophosphorylation sites on EGFR, did depend on the identity of the kinase domain. Our data also showed that ligand dependence was lost when the native kinase domains were replaced by Src, suggesting that the identity of the kinase domains could be important for proper receptor regulation. Overall, the results are consistent with the idea that the fidelity of RTK signaling depends on co-localization and targeting with substrates, as well as on the intrinsic specificity of the kinase domain.

CD248 promotes insulin resistance by binding to the insulin receptor and dampening its insulin-induced autophosphorylation.

BACKGROUND: In spite of new treatments, the incidence of type 2 diabetes (T2D) and its morbidities continue to rise. The key feature of T2D is resistance of adipose tissue and other organs to insulin. Approaches to overcome insulin resistance are limited due to a poor understanding of the mechanisms and inaccessibility of drugs to relevant intracellular targets. We previously showed in mice and humans that CD248, a pre/adipocyte cell surface glycoprotein, acts as an adipose tissue sensor that mediates the transition from healthy to unhealthy adipose, thus promoting insulin resistance. METHODS: Molecular mechanisms by which CD248 regulates insulin signaling were explored using in vivo insulin clamp studies and biochemical analyses of cells/tissues from CD248 knockout (KO) and wild-type (WT) mice with diet-induced insulin resistance. Findings were validated with human adipose tissue specimens. FINDINGS: Genetic deletion of CD248 in mice, overcame diet-induced insulin resistance with improvements in glucose uptake and lipolysis in white adipose tissue depots, effects paralleled by increased adipose/adipocyte GLUT4, phosphorylated AKT and GSK3ß, and reduced ATGL. The insulin resistance of the WT mice could be attributed to direct interaction of the extracellular domains of CD248 and the insulin receptor (IR), with CD248 acting to block insulin binding to the IR. This resulted in dampened insulin-mediated autophosphorylation of the IR, with reduced downstream signaling/activation of intracellular events necessary for glucose and lipid homeostasis. INTERPRETATION: Our discovery of a cell-surface CD248-IR complex that is accessible to pharmacologic intervention, opens research avenues toward development of new agents to prevent/reverse insulin resistance. FUNDING: Funded by Canadian Institutes of Health Research (CIHR), Natural Sciences and Engineering Research Council of Canada (NSERC), Canada Foundations for Innovation (CFI), the Swedish Diabetes Foundation, Family Ernfors Foundation and Novo Nordisk Foundation.

A microglia clonal inflammatory disorder in Alzheimer's Disease.

Somatic genetic heterogeneity resulting from post-zygotic DNA mutations is widespread in human tissues and can cause diseases, however few studies have investigated its role in neurodegenerative processes such as Alzheimer's Disease (AD). Here we report the selective enrichment of microglia clones carrying pathogenic variants, that are not present in neuronal, glia/stromal cells, or blood, from patients with AD in comparison to age-matched controls. Notably, microglia-specific AD-associated variants preferentially target the MAPK pathway, including recurrent CBL ring-domain mutations. These variants activate ERK and drive a microglia transcriptional program characterized by a strong neuro-inflammatory response, both in vitro and in patients. Although the natural history of AD-associated microglial clones is difficult to establish in human, microglial expression of a MAPK pathway activating variant was previously shown to cause neurodegeneration in mice, suggesting that AD-associated neuroinflammatory microglial clones may contribute to the neurodegenerative process in patients.

Metazoan-like signaling in a unicellular receptor tyrosine kinase.

BACKGROUND: Receptor tyrosine kinases (RTKs) are crucial components of signal transduction systems in multicellular animals. Surprisingly, numerous RTKs have been identified in the genomes of unicellular choanoflagellates and other protists. Here, we report the first biochemical study of a unicellular RTK, namely RTKB2 from Monosiga brevicollis. RESULTS: We cloned, expressed, and purified the RTKB2 kinase, and showed that it is enzymatically active. The activity of RTKB2 is controlled by autophosphorylation, as in metazoan RTKs. RTKB2 possesses six copies of a unique domain (designated RM2) in its C-terminal tail. An isolated RM2 domain (or a synthetic peptide derived from the RM2 sequence) served as a substrate for RTKB2 kinase. When phosphorylated, the RM2 domain bound to the Src homology 2 domain of MbSrc1 from M. brevicollis. NMR structural studies of the RM2 domain indicated that it is disordered in solution. CONCLUSIONS: Our results are consistent with a model in which RTKB2 activation stimulates receptor autophosphorylation within the RM2 domains. This leads to recruitment of Src-like kinases (and potentially other M. brevicollis proteins) and further phosphorylation, which may serve to increase or dampen downstream signals. Thus, crucial features of signal transduction circuitry were established prior to the evolution of metazoans from their unicellular ancestors.

Phosphorylation of Ack1 by the Receptor Tyrosine Kinase Mer.

Ack1 is a nonreceptor tyrosine kinase that is associated with cellular proliferation and survival. The receptor tyrosine kinase Mer, a member of the TAM family of receptors, has previously been reported to be an upstream activator of Ack1 kinase. The mechanism linking the two kinases, however, has not been investigated. We confirmed that Ack1 and Mer interact by co-immunoprecipitation experiments and found that Mer expression led to increased Ack1 activity. The effect on Ack1 was dependent on the kinase activity of Mer, whereas mutation of the Mer C-terminal tyrosines Y867 and Y924 did not significantly decrease the ability of Mer to activate Ack1. Ack1 possesses a Mig6 Homology Region (MHR) that contains adjacent regulatory tyrosines (Y859 and Y860). Using synthetic peptides, we showed that Mer preferentially binds and phosphorylates the MHR sequence containing phosphorylated pY860, as compared to the pY859 sequence. This suggested the possibility of sequential phosphorylation within the MHR of Ack1, as has been observed previously for other kinases. In cells co-expressing Mer and Ack1 MHR mutants, the Y859F mutant had higher activity than the Y860F mutant, consistent with this model. The interaction between Mer and Ack1 could play a role in immune cell signaling in normal physiology and could also contribute to the hyperactivation of Ack1 in prostate cancer and other tumors.

Small-molecule inhibition and activation-loop trans-phosphorylation of the IGF1 receptor.

The insulin-like growth factor-1 receptor (IGF1R) is a receptor tyrosine kinase (RTK) that has a critical role in mitogenic signalling during embryogenesis and an antiapoptotic role in the survival and progression of many human tumours. Here, we present the crystal structure of the tyrosine kinase domain of IGF1R (IGF1RK), in its unphosphorylated state, in complex with a novel compound, cis-3-[3-(4-methyl-piperazin-l-yl)-cyclobutyl]-1-(2-phenyl-quinolin-7-yl)-imidazo[1,5-a]pyrazin-8-ylamine (PQIP), which we show is a potent inhibitor of both the unphosphorylated (basal) and phosphorylated (activated) states of the kinase. PQIP interacts with residues in the ATP-binding pocket and in the activation loop, which confers specificity for IGF1RK and the highly related insulin receptor (IR) kinase. In this crystal structure, the IGF1RK active site is occupied by Tyr1135 from the activation loop of an symmetry (two-fold)-related molecule. This dimeric arrangement affords, for the first time, a visualization of the initial trans-phosphorylation event in the activation loop of an RTK, and provides a molecular rationale for a naturally occurring mutation in the activation loop of the IR that causes type II diabetes mellitus.

Cancer-associated mutations activate the nonreceptor tyrosine kinase Ack1.

Ack1 is a nonreceptor tyrosine kinase that participates in tumorigenesis, cell survival, and migration. Relatively little is known about the mechanisms that regulate Ack1 activity. Recently, four somatic missense mutations of Ack1 were identified in cancer tissue samples, but the effects on Ack1 activity, and function have not been described. These mutations occur in the N-terminal region, the C-lobe of the kinase domain, and the SH3 domain. Here, we show that the cancer-associated mutations increase Ack1 autophosphorylation in mammalian cells without affecting localization and increase Ack1 activity in immune complex kinase assays. The cancer-associated mutations potentiate the ability of Ack1 to promote proliferation and migration, suggesting that point mutation is a mechanism for Ack1 deregulation. We propose that the C-terminal Mig6 homology region (MHR) (residues 802-990) participates in inhibitory intramolecular interactions. The isolated kinase domain of Ack1 interacts directly with the MHR, and the cancer-associated E346K mutation prevents binding. Likewise, mutation of a key hydrophobic residue in the MHR (Phe(820)) prevents the MHR-kinase interaction, activates Ack1, and increases cell migration. Thus, the cancer-associated mutation E346K appears to destabilize an autoinhibited conformation of Ack1, leading to constitutively high Ack1 activity.

Substrate binding to Src: A new perspective on tyrosine kinase substrate recognition from NMR and molecular dynamics.

Most signal transduction pathways in humans are regulated by protein kinases through phosphorylation of their protein substrates. Typical eukaryotic protein kinases are of two major types: those that phosphorylate-specific sequences containing tyrosine (~90 kinases) and those that phosphorylate either serine or threonine (~395 kinases). The highly conserved catalytic domain of protein kinases comprises a smaller N lobe and a larger C lobe separated by a cleft region lined by the activation loop. Prior studies find that protein tyrosine kinases recognize peptide substrates by binding the polypeptide chain along the C-lobe on one side of the activation loop, while serine/threonine kinases bind their substrates in the cleft and on the side of the activation loop opposite to that of the tyrosine kinases. Substrate binding structural studies have been limited to four families of the tyrosine kinase group, and did not include Src tyrosine kinases. We examined peptide-substrate binding to Src using paramagnetic-relaxation-enhancement NMR combined with molecular dynamics simulations. The results suggest Src tyrosine kinase can bind substrate positioning residues C-terminal to the phosphoacceptor residue in an orientation similar to serine/threonine kinases, and unlike other tyrosine kinases. Mutagenesis corroborates this new perspective on tyrosine kinase substrate recognition. Rather than an evolutionary split between tyrosine and serine/threonine kinases, a change in substrate recognition may have occurred within the TK group of the human kinome. Protein tyrosine kinases have long been therapeutic targets, but many marketed drugs have deleterious off-target effects. More accurate knowledge of substrate interactions of tyrosine kinases has the potential for improving drug selectivity.

Reengineering the signaling properties of a Src family kinase.

Src family kinases (SFKs) are modular signaling proteins possessing SH3, SH2, and tyrosine kinase domains. The SH3 and SH2 domains of SFKs have dual roles: they regulate the activity of the kinases, and they also target SFKs to their cellular substrates. We generated a series of novel SFKs by replacing the SH2 and SH3 domains of Hck with the syntrophin PDZ domain. In some constructs, the negative regulatory tyrosine in the C-terminal tail was also replaced with a PDZ ligand sequence. When expressed in mammalian cells, the substrate specificity of the PDZ-kinases was directed to a different group of proteins than wild-type Hck. The PDZ-kinases phosphorylate neuronal nitric oxide synthase (nNOS), a known binding partner of the syntrophin PDZ domain. We also introduced a PDZ ligand at the C-terminus of the adaptor protein Cas. PDZ-Hck kinases phosphorylate the engineered Cas protein in Cas(-/-) cells and restore the migration defect of these cells. A PDZ-kinase was also functional in rewiring MAPK signaling via an engineered ErbB2 construct containing a PDZ ligand sequence. Several of the PDZ-kinases show autoregulatory properties similar to natural SFKs. Thus, the PDZ-ligand interaction is able to functionally replace the normal SH2-pY527 interaction that regulates SFKs. Our data highlight the modularity and evolvability of signaling proteins.

Autoinhibition of the insulin-like growth factor I receptor by the juxtamembrane region.

The juxtamembrane (JM) regions of several receptor tyrosine kinases are involved in autoinhibitory interactions that maintain the low basal activity of the receptors; mutations can give rise to constitutive kinase activity and signaling. In this report, we show that the JM region of the human insulin-like growth factor I receptor (IGF1R) plays a role in kinase regulation. We mutated JM residues that were conserved in this subfamily of receptor tyrosine kinases, and expressed and purified the cytoplasmic domains using the Sf9/baculovirus system. We show that a kinase-proximal mutation (Y957F) and (to a lesser extent) a mutation in the central part of the JM region (N947A) increase the autophosphorylation activity of the kinase. Steady-state kinetic measurements show the mutations cause an increase in V(max) for phosphorylation of peptide substrates. When the holoreceptors were expressed in fibroblasts derived from IGF1R-deficient mice, the Y957F mutation led to a large increase in basal and in IGF1-stimulated receptor autophosphorylation. Together, these data demonstrate that the JM region of IGF1R plays an important role in limiting the basal activity of the receptor.

Structural and Biochemical Basis for Intracellular Kinase Inhibition by Src-specific Peptidic Macrocycles.

Protein kinases are attractive therapeutic targets because their dysregulation underlies many diseases, including cancer. The high conservation of the kinase domain and the evolution of drug resistance, however, pose major challenges to the development of specific kinase inhibitors. We recently discovered selective Src kinase inhibitors from a DNA-templated macrocycle library. Here, we reveal the structural basis for how these inhibitors retain activity against a disease-relevant, drug-resistant kinase mutant, while maintaining Src specificity. We find that these macrocycles display a degree of modularity: two of their three variable groups interact with sites on the kinase that confer selectivity, while the third group interacts with the universally conserved catalytic lysine and thereby retains the ability to inhibit the "gatekeeper" kinase mutant. We also show that these macrocycles inhibit migration of MDA-MB-231 breast tumor cells. Our findings establish intracellular kinase inhibition by peptidic macrocycles, and inform the development of potent and specific kinase inhibitors.

Constitutive Activity in an Ancestral Form of Abl Tyrosine Kinase.

The c-abl proto-oncogene encodes a nonreceptor tyrosine kinase that is found in all metazoans, and is ubiquitously expressed in mammalian tissues. The Abl tyrosine kinase plays important roles in the regulation of mammalian cell physiology. Abl-like kinases have been identified in the genomes of unicellular choanoflagellates, the closest relatives to the Metazoa, and in related unicellular organisms. Here, we have carried out the first characterization of a premetazoan Abl kinase, MbAbl2, from the choanoflagellate Monosiga brevicollis. The enzyme possesses SH3, SH2, and kinase domains in a similar arrangement to its mammalian counterparts, and is an active tyrosine kinase. MbAbl2 lacks the N-terminal myristoylation and cap sequences that are critical regulators of mammalian Abl kinase activity, and we show that MbAbl2 is constitutively active. When expressed in mammalian cells, MbAbl2 strongly phosphorylates cellular proteins on tyrosine, and transforms cells much more potently than mammalian Abl kinase. Thus, MbAbl2 appears to lack the autoinhibitory mechanism that tightly constrains the activity of mammalian Abl kinases, suggesting that this regulatory apparatus arose more recently in metazoan evolution.

Effects of somatic mutations in the C-terminus of insulin-like growth factor 1 receptor on activity and signaling.

The insulin-like growth factor I receptor (IGF1R) is overexpressed in several forms of human cancer, and it has emerged as an important target for anticancer drug design. Cancer genome sequencing efforts have recently identified three somatic mutations in IGF1R: A1374V, a deletion of S1278 in the C-terminal tail region of the receptor, and M1255I in the C-terminal lobe of the kinase catalytic domain. The possible effects of these mutations on IGF1R activity and biological function have not previously been tested. Here, we tested the effects of the mutations on the in vitro biochemical activity of IGF1R and on major IGF1R signaling pathways in mammalian cells. While the mutations do not affect the intrinsic tyrosine kinase activity of the receptor, we demonstrate that the basal (unstimulated) levels of MAP kinase and Akt activation are increased in the mutants (relative to wild-type IGF1R). We hypothesize that the enhanced signaling potential of these mutants is due to changes in protein-protein interactions between the IGF1R C-terminus and cellular substrates or modulators.

PTB domain-directed substrate targeting in a tyrosine kinase from the unicellular choanoflagellate Monosiga brevicollis.

Choanoflagellates are considered to be the closest living unicellular relatives of metazoans. The genome of the choanoflagellate Monosiga brevicollis contains a surprisingly high number and diversity of tyrosine kinases, tyrosine phosphatases, and phosphotyrosine-binding domains. Many of the tyrosine kinases possess combinations of domains that have not been observed in any multicellular organism. The role of these protein interaction domains in M. brevicollis kinase signaling is not clear. Here, we have carried out a biochemical characterization of Monosiga HMTK1, a protein containing a putative PTB domain linked to a tyrosine kinase catalytic domain. We cloned, expressed, and purified HMTK1, and we demonstrated that it possesses tyrosine kinase activity. We used immobilized peptide arrays to define a preferred ligand for the third PTB domain of HMTK1. Peptide sequences containing this ligand sequence are phosphorylated efficiently by recombinant HMTK1, suggesting that the PTB domain of HMTK1 has a role in substrate recognition analogous to the SH2 and SH3 domains of mammalian Src family kinases. We suggest that the substrate recruitment function of the noncatalytic domains of tyrosine kinases arose before their roles in autoinhibition.

Synthesis of functional signaling domains by combinatorial polymerization of phosphorylation motifs.

The adaptor protein Cas contains a core substrate domain with multiple YXXP motifs that are phosphorylated by Src and other tyrosine kinases. Here, we used a synthetic strategy to determine the importance of the arrangement, spacing, and identity of the YXXP motifs. By polymerizing short DNA sequences encoding two phosphorylation motifs, we created a panel of Cas mutants in which the entire substrate domain was replaced by synthetic domains containing random numbers and arrangements of the motifs. Most of these synthetic Cas variants were recognized and phosphorylated by Src in vitro and in intact mammalian cells. The random polymer mutants also restored migration activity to Cas knockout cells; even artificial proteins containing a single motif retained some biological function. Our results suggest that the arrangement of Cas motifs is not critical for signaling. This method could be used to identify the minimal functional units in other signaling proteins.