Stimulation Of Kinase Activity Research Articles

Investigating the Neuroprotective and Cognitive-Enhancing Effects of Bacopa monnieri: A Systematic Review Focused on Inflammation, Oxidative Stress, Mitochondrial Dysfunction, and Apoptosis.

The aging of the global population has increased the prevalence of neurodegenerative conditions. Bacopa monnieri (BM), an herb with active compounds, such as bacosides A and B, betulinic acid, loliolide, asiatic acid, and quercetin, demonstrates the potential for brain health. Limited research has been conducted on the therapeutic applications of BM in neurodegenerative conditions. This systematic review aims to project BM's beneficial role in brain disorders. BM has anti-apoptotic and antioxidant actions and can repair damaged neurons, stimulate kinase activity, restore synaptic function, improve nerve transmission, and increase neuroprotection. The included twenty-two clinical trials demonstrated that BM can reduce Nuclear Factor-κB phosphorylation, improve emotional function, cognitive functions, anhedonia, hyperactivity, sleep routine, depression, attention deficit, learning problems, memory retention, impulsivity, and psychiatric problems. Moreover, BM can reduce the levels of pro-inflammatory biomarkers and oxidative stress. Here, we highlight that BM provides notable therapeutic benefits and can serve as a complementary approach for the care of patients with neurodegenerative conditions associated with brain disorders. This review adds to the growing interest in natural products and their potential therapeutic applications by improving our understanding of the mechanisms underlying cognitive function and neurodegeneration and informing the development of new therapeutic strategies for neurodegenerative diseases.

Drug repurposing approach for identifying Pfmrk inhibitors as potential antimalarial agents: An in silico analysis

Malaria represents a major global health concern, primarily due to the emergence of resistance against most currently available antimalarial drugs. This pressing issue necessitates the discovery of novel antimalarial agents to combat the escalating resistance. A cyclin-dependent kinase (CDK)-like protein, Pfmrk, found in Plasmodium falciparum, plays a crucial role in regulating cell proliferation and exhibits a 36.28% sequence homology with its human counterpart hCDK7. Pfmrk forms a complex with plasmodial cyclin (Pfcyc-1) and stimulates kinase activity. Pfcyc-1 from P. falciparum, with the highest sequence homology to human cyclin (cyclin H), binds and activates Pfmrk in a cyclin-dependent manner. This discovery provides the first indication that cyclin subunits may regulate both human and plasmodial CDKs in a similar fashion. In this study, we conducted molecular docking and simulation analysis to investigate the interaction between Pfmrk and a selection of the FDA-approved drugs retrieved from the ZINC15 database. The top five drugs – Lurasidone, Vorapaxar, Donovex, Alvesco, and Orap – were screened based on their binding energies, with the best-docked scores ranging between −8 kcal/mol and −12 kcal/mol. Further, evaluation through molecular dynamics simulations for 100 nanoseconds revealed that Lurasidone exhibited the highest binding affinity (−105.90 ± 57.72 kJ/mol) followed by Donovex (−92.877 ± 17.872 kJ/mol). They exhibited stable interactions with the amino acid residues located in the active site of Pfmrk. The results of the in silico investigation indicate that Lurasidone and Donovex exhibit antimalarial potential and could serve as promising Pfmrk inhibitors. Further, development of new drugs based on these findings warrants subsequent in vitro studies.

Impact of 100 LRRK2 variants linked to Parkinson's disease on kinase activity and microtubule binding.

Mutations enhancing the kinase activity of leucine-rich repeat kinase-2 (LRRK2) cause Parkinson's disease (PD) and therapies that reduce LRRK2 kinase activity are being tested in clinical trials. Numerous rare variants of unknown clinical significance have been reported, but how the vast majority impact on LRRK2 function is unknown. Here, we investigate 100 LRRK2 variants linked to PD, including previously described pathogenic mutations. We identify 23 LRRK2 variants that robustly stimulate kinase activity, including variants within the N-terminal non-catalytic regions (ARM (E334K, A419V), ANK (R767H), LRR (R1067Q, R1325Q)), as well as variants predicted to destabilize the ROC:CORB interface (ROC (A1442P, V1447M), CORA (R1628P) CORB (S1761R, L1795F)) and COR:COR dimer interface (CORB (R1728H/L)). Most activating variants decrease LRRK2 biomarker site phosphorylation (pSer935/pSer955/pSer973), consistent with the notion that the active kinase conformation blocks their phosphorylation. We conclude that the impact of variants on kinase activity is best evaluated by deploying a cellular assay of LRRK2-dependent Rab10 substrate phosphorylation, compared with a biochemical kinase assay, as only a minority of activating variants (CORB (Y1699C, R1728H/L, S1761R) and kinase (G2019S, I2020T, T2031S)), enhance in vitro kinase activity of immunoprecipitated LRRK2. Twelve variants including several that activate LRRK2 and have been linked to PD, suppress microtubule association in the presence of a Type I kinase inhibitor (ARM (M712V), LRR (R1320S), ROC (A1442P, K1468E, S1508R), CORA (A1589S), CORB (Y1699C, R1728H/L) and WD40 (R2143M, S2350I, G2385R)). Our findings will stimulate work to better understand the mechanisms by which variants impact biology and provide rationale for variant carrier inclusion or exclusion in ongoing and future LRRK2 inhibitor clinical trials.

Role of Ca2+/Calmodulin-Dependent Protein Kinase Type II in Mediating Function and Dysfunction at Glutamatergic Synapses.

Glutamatergic synapses harbor abundant amounts of the multifunctional Ca2+/calmodulin-dependent protein kinase type II (CaMKII). Both in the postsynaptic density as well as in the cytosolic compartment of postsynaptic terminals, CaMKII plays major roles. In addition to its Ca2+-stimulated kinase activity, it can also bind to a variety of membrane proteins at the synapse and thus exert spatially restricted activity. The abundance of CaMKII in glutamatergic synapse is akin to scaffolding proteins although its prominent function still appears to be that of a kinase. The multimeric structure of CaMKII also confers several functional capabilities on the enzyme. The versatility of the enzyme has prompted hypotheses proposing several roles for the enzyme such as Ca2+ signal transduction, memory molecule function and scaffolding. The article will review the multiple roles played by CaMKII in glutamatergic synapses and how they are affected in disease conditions.

Imatinib disassembles the regulatory core of Abelson kinase by binding to its ATP site and not by binding to its myristoyl pocket.

It was recently reported (Xie et al., 2022) that the Abelson tyrosine kinase (Abl) ATP-site inhibitor imatinib also binds to Abl's myristoyl binding pocket, which is the target of allosteric Abl inhibitors. This was based on a crystal structure of a truncated Abl kinase domain construct in complex with imatinib bound to the allosteric site as well as further isothermal titration calorimetry (ITC), NMR, and kinase activity data. Although imatinib's affinity for the allosteric site is significantly weaker (10 M) than for the ATP site (10 nM), imatinib binding to the allosteric site may disassemble the regulatory core of Abl, thereby stimulating kinase activity, in particular for Abl mutants with reduced imatinib ATP-site affinity. It was argued that the previously observed imatinib-induced opening of the Abl regulatory core (Skora et al., 2013; Sonti et al., 2018) may be caused by the binding of imatinib to the allosteric site and not to the ATP site. We show here that this is not the case but that indeed imatinib binding to the ATP site induces the opening of the regulatory core at nanomolar concentrations. This agrees with findings that other type-II ATP-site inhibitors (nilotinib, ponatinib) disassemble the regulatory core despite demonstrated negligible binding to the allosteric site.

Characterization of six CaMKIIα variants found in patients with schizophrenia

SummaryThe Ca2+/Calmodulin-dependent protein kinase II (CaMKII) is a central regulator of synaptic plasticity and has been implicated in various neurological conditions, including schizophrenia. Here, we characterize six different CaMKIIα variants found in patients with schizophrenia. Only R396stop disrupted the 12-meric holoenzyme structure, GluN2B binding, and synaptic localization. Additionally, R396stop impaired T286 autophosphorylation that generates Ca2+-independent “autonomous” kinase activity. This impairment in T286 autophosphorylation was shared by the R8H mutation, the only mutation that additionally reduced stimulated kinase activity. None of the mutations affected the levels of CaMKII expression in HEK293 cells. Thus, impaired CaMKII function was detected only for R396stop and R8H. However, two of the other mutations have been later identified also in the general population, and not all mutations found in patients with schizophrenia would be expected to cause disease. Nonetheless, for the R396stop mutation, the severity of the biochemical effects found here would predict a neurological phenotype.

Immune Sensing of Cell Death through Recognition of Histone Sequences by C-Type Lectin-Receptor-2d Causes Inflammation and Tissue Injury

The immune system monitors the health of cells and is stimulated by necrosis. Here we examined the receptors and ligands driving this response. In a targeted screen of C-type lectin receptors, a Clec2d reporter responded to lysates from necrotic cells. Biochemical purification identified histones, both free and bound to nucleosomes or neutrophil extracellular traps, as Clec2d ligands. Clec2d recognized poly-basic sequences in histone tails and this recognition was sensitive to post-translational modifications of these sequences. As compared with WT mice, Clec2d-/- mice exhibited reduced proinflammatory responses to injected histones, and less tissue damage and improved survival in a hepatotoxic injury model. In macrophages, Clec2d localized to the plasma membrane and endosomes. Histone binding to Clec2d did not stimulate kinase activation or cytokine production. Rather, histone-bound DNA stimulated endosomal Tlr9-dependent responses in a Clec2d-dependent manner. Thus, Clec2d binds to histones released upon necrotic cell death, with functional consequences to inflammation and tissue damage.

Ddc2ATRIP promotes Mec1ATR activation at RPA-ssDNA tracts.

The DNA damage checkpoint response is controlled by the phosphatidylinositol 3-kinase-related kinases (PIKK), including ataxia telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR). ATR forms a complex with its partner ATRIP. In budding yeast, ATR and ATRIP correspond to Mec1 and Ddc2, respectively. ATRIP/Ddc2 interacts with replication protein A-bound single-stranded DNA (RPA-ssDNA) and recruits ATR/Mec1 to sites of DNA damage. Mec1 is stimulated by the canonical activators including Ddc1, Dpb11 and Dna2. We have characterized the ddc2-S4 mutation and shown that Ddc2 not only recruits Mec1 to sites of DNA damage but also stimulates Mec1 kinase activity. However, the underlying mechanism of Ddc2-dependent Mec1 activation remains to be elucidated. Here we show that Ddc2 promotes Mec1 activation independently of Ddc1/Dpb11/Dna2 function in vivo and through ssDNA recognition in vitro. The ddc2-S4 mutation diminishes damage-induced phosphorylation of the checkpoint mediators, Rad9 and Mrc1. Rad9 controls checkpoint throughout the cell-cycle whereas Mrc1 is specifically required for the S-phase checkpoint. Notably, S-phase checkpoint signaling is more defective in ddc2-S4 mutants than in cells where the Mec1 activators (Ddc1/Dpb11 and Dna2) are dysfunctional. To understand a role of Ddc2 in Mec1 activation, we reconstituted an in vitro assay using purified Mec1-Ddc2 complex, RPA and ssDNA. Whereas ssDNA stimulates kinase activity of the Mec1-Ddc2 complex, RPA does not. However, RPA can promote ssDNA-dependent Mec1 activation. Neither ssDNA nor RPA-ssDNA efficiently stimulates the Mec1-Ddc2 complex containing Ddc2-S4 mutant. Together, our data support a model in which Ddc2 promotes Mec1 activation at RPA-ssDNA tracts.

Asterless is a Polo-like kinase 4 substrate that both activates and inhibits kinase activity depending on its phosphorylation state.

Centriole assembly initiates when Polo-like kinase 4 (Plk4) interacts with a centriole “targeting-factor.” In Drosophila, Asterless/Asl (Cep152 in humans) fulfills the targeting role. Interestingly, Asl also regulates Plk4 levels. The N-terminus of Asl (Asl-A; amino acids 1-374) binds Plk4 and promotes Plk4 self-destruction, although it is unclear how this is achieved. Moreover, Plk4 phosphorylates the Cep152 N-terminus, but the functional consequence is unknown. Here, we show that Plk4 phosphorylates Asl and mapped 13 phospho-residues in Asl-A. Nonphosphorylatable alanine (13A) and phosphomimetic (13PM) mutants did not alter Asl function, presumably because of the dominant role of the Asl C-terminus in Plk4 stabilization and centriolar targeting. To address how Asl-A phosphorylation specifically affects Plk4 regulation, we generated Asl-A fragment phospho-mutants and expressed them in cultured Drosophila cells. Asl-A-13A stimulated kinase activity by relieving Plk4 autoinhibition. In contrast, Asl-A-13PM inhibited Plk4 activity by a novel mechanism involving autophosphorylation of Plk4’s kinase domain. Thus, Asl-A’s phosphorylation state determines which of Asl-A’s two opposing effects are exerted on Plk4. Initially, nonphosphorylated Asl binds Plk4 and stimulates its kinase activity, but after Asl is phosphorylated, a negative-feedback mechanism suppresses Plk4 activity. This dual regulatory effect by Asl-A may limit Plk4 to bursts of activity that modulate centriole duplication.

The essential kinase ATR: ensuring faithful duplication of a challenging genome.

One way to preserve a rare book is to lock it away from all potential sources of damage. Of course, an inaccessible book is also of little use, and the paper and ink will continue to degrade with age in any case. Like a book, the information stored in our DNA needs to be read, but it is also subject to continuous assault and therefore needs to be protected. In this Review, we examine how the replication stress response that is controlled by the kinase ataxia telangiectasia and Rad3-related (ATR) senses and resolves threats to DNA integrity so that the DNA remains available to read in all of our cells. We discuss the multiple data that have revealed an elegant yet increasingly complex mechanism of ATR activation. This involves a core set of components that recruit ATR to stressed replication forks, stimulate kinase activity and amplify ATR signalling. We focus on the activities of ATR in the control of cell cycle checkpoints, origin firing and replication fork stability, and on how proper regulation of these processes is crucial to ensure faithful duplication of a challenging genome.

Structure of the Shroom-Rho Kinase Complex Reveals a Binding Interface with Monomeric Shroom That Regulates Cell Morphology and Stimulates Kinase Activity

Shroom-mediated remodeling of the actomyosin cytoskeleton is a critical driver of cellular shape and tissue morphology that underlies the development of many tissues including the neural tube, eye, intestines, and vasculature. Shroom uses a conserved SD2 domain to direct the subcellular localization of Rho-associated kinase (Rock), which in turn drives changes in the cytoskeleton and cellular morphology through its ability to phosphorylate and activate non-muscle myosin II. Here, we present the structure of the human Shroom-Rock binding module, revealing an unexpected stoichiometry for Shroom in which two Shroom SD2 domains bind independent surfaces on Rock. Mutation of interfacial residues impaired Shroom-Rock binding in vitro and resulted in altered remodeling of the cytoskeleton and loss of Shroom-mediated changes in cellular morphology. Additionally, we provide the first direct evidence that Shroom can function as a Rock activator. These data provide molecular insight into the Shroom-Rock interface and demonstrate that Shroom directly participates in regulating cytoskeletal dynamics, adding to its known role in Rock localization.

68 - Redox Regulation of ASK1 by Peroxiredoxin 1 and Thioredoxin 1

Apoptosis signal-regulating kinase 1 (ASK1) is a redox sensitive kinase that is critical in cell stress signalling. The mechanism by which redox stress alters ASK1 kinase activity remains controversial. Early data suggested that binding of reduced thioredoxin 1 (Trx1) to ASK1 inhibited activity, and dissociation of oxidised Trx1 relieved the inhibition. Subsequently it has been reported that oxidation of ASK1 itself stimulates kinase activity and we have previously shown that peroxiredoxin 1 (Prdx1) can transmit a H2O2 signal to ASK1 via disulfide bond-dependent oligomerization and activation of ASK11. Thus the relationship between ASK1 oxidation, Trx1 binding and kinase activity remain unclear. Here we use recombinant wild-type and cysteine mutants of ASK1, Prdx1 and Trx to reconstitute redox regulation of ASK1 in vitro. Our aims are to identify which ASK1 cysteine residues are important for interaction of ASK1 with Prdx1 and Trx1, how oxidation of ASK1 alters its kinase activity. Using wild-type Trx1 and a Trx1 trapping mutant (C35S) we find that Trx1 interacts both covalently and non-covalently with ASK1. We have purified a covalently-linked ASK1-Trx1 (C35S) complex and using LC-MS/MS we have identified cysteine residues within the N-terminal regulatory domain of ASK1 that are required for disulfide formation with Trx1. This result has been confirmed by mutagenesis studies. We conclude that the N-terminal regulatory domain is the main target for redox regulation of ASK-1, and that ASK1 is a substrate for the oxidoreductase activity of Trx1. 1Jarvis et al (2012) Free Radic Biol Med 53, 1522-1530

Crystal structure of an SH2-kinase construct of c-Abl and effect of the SH2 domain on kinase activity.

Constitutive activation of the non-receptor tyrosine kinase c-Abl (cellular Abelson tyrosine protein kinase 1, Abl1) in the Bcr (breakpoint cluster region)-Abl1 fusion oncoprotein is the molecular cause of chronic myeloid leukaemia (CML). Recent studies have indicated that an interaction between the SH2 (Src-homology 2) domain and the N-lobe (N-terminal lobe) of the c-Abl kinase domain (KD) has a critical role in leukaemogenesis [Grebien et al. (2011) Cell 147, 306-319; Sherbenou et al. (2010) Blood 116, 3278-3285]. To dissect the structural basis of this phenomenon, we studied c-Abl constructs comprising the SH2 and KDs invitro. We present a crystal structure of an SH2-KD construct bound to dasatinib, which contains the relevant interface between the SH2 domain and the N-lobe of the KD. We show that the presence of the SH2 domain enhances kinase activity moderately and that this effect depends on contacts in the SH2/N-lobe interface and is abrogated by specific mutations. Consistently, formation of the interface decreases slightly the association rate of imatinib with the KD. That the effects are small compared with the dramatic invivo consequences suggests an important function of the SH2-N-lobe interaction might be to help disassemble the auto-inhibited conformation of c-Abl and promote processive phosphorylation, rather than substantially stimulate kinase activity.

Inhibiting Eph kinase activity may not be “Eph”ective for cancer treatment

Several Eph receptor tyrosine kinases (RTKs) are commonly over-expressed in epithelial and mesenchymal cancers and are recognized as promising therapeutic targets. Although normal interaction between Eph receptors and their ephrin ligands stimulates kinase activity and is generally tumor suppressive, significant Eph over-expression allows activation of ligand- and/or kinase-independent signaling pathways that promote oncogenesis. Single-agent kinase inhibitors are widely used to target RTK-driven tumors but acquired and de novo resistance to such agents is a major limitation to effective clinical use. Accumulating evidence suggests that Ephs can be inhibited by “leaky” or low-specificity kinase inhibitors targeted at other RTKs. Such off-target effects may therefore inadvertently promote ligand- and/or kinase-independent oncogenic Eph signaling, thereby providing a new mechanism by which resistance to the RTK inhibitors can emerge. We propose that combining specific, non-leaky kinase inhibitors with tumor-suppressive stimulators of Eph signaling may provide more effective treatment options for overcoming treatment-induced resistance and clinical failure.

C-Abl Activates Janus Kinase 2 in Normal Hematopoietic Cells

Jak2 is involved in cytokine growth factor-stimulated signal transduction, but the mechanism of its activation is largely unknown. Here, we investigated Jak2 activation in a normal hematopoietic cell line, 32D mouse myeloid cells. The bimolecular fluorescence complementation studies showed that c-Abl formed a stable complex with Jak2 in live cells. Co-immunoprecipitation results showed that c-Abl bound to the βc chain of IL-3/IL-5/GM-CSF receptors. The kinase activities of both c-Abl and Jak2 were stimulated by IL-3 in 32D cells. Decreasing c-Abl protein expression in 32D cells by inducible shRNA decreased Jak2 activity and resulted in the failure of Jak2 activation in response to IL-3. Treatment of IL-3 and serum-starved 32D cells with 1 μM imatinib mysylate inhibited IL-3 stimulated kinase activities of both c-Abl and Jak2. In addition, the kinase-deficient Bcr-Abl mutant (p210K1172R) was defective for activation of Jak2 in 32D cells and impaired IL-3 independent growth, which was rescued by overexpression of c-Abl (+Abl). IL-3 efficiently inhibited apoptosis of 32Dp210K/R+Abl cells induced by imatinib mysylate but not Jak2 kinase inhibitor TG101209. In summary, our findings provide evidence that the kinase function of c-Abl and its C-terminal CT4 region is crucial for its interaction with Jak2 and its activation. c-Abl kinase activity induced by IL-3 is required for IL-3-stimulated Jak2 and Jak1 activation. Our findings reveal a novel regulatory role of c-Abl in Jak2 activation induced by IL-3 cytokine growth factor in 32D hematopoietic cells.

Mutation of mpk6 enhances cadmium tolerance in Arabidopsis plants by alleviating oxidative stress

Cadmium (Cd) could activate activity of mitogen-activated protein kinase MPK6 in plants. In this study, we investigated the role of MPK6 in mediating Cd toxicity in plants. The wild type Arabidopsis plants (WT) and the mpk6-2 mutants were subjected either 0 (Control) or 10 μM Cd treatment. Kinase activity of MPK6, nitric oxide (NO) level, Cd concentration, and oxidative stress were measured. In WT plants, Cd exposure rapidly stimulated kinase activity of MPK6. However, upon Cd exposure, mpk6-2 showed better growth than the WT. Although Cd-induced production of NO in roots was greater in WT than in mpk6-2, there was no difference in Cd concentration between the two plants. Nevertheless, the Cd-induced hydroperoxide burst, lipid peroxidation and loss of membrane integrity, were all more severe in the WT than in mpk6-2. Foliar applications of antioxidant ascorbic acid, vigorously improved the growth of both the WT and mpk6-2 under Cd exposure. Thereby the growth difference between these two plants was minimized. Mutation of mpk6 enhances Cd tolerance in plants by alleviating oxidative stress, but did not affect cadmium accumulation in plants.

HSF1, A Versatile Factor in Tumorogenesis

HSF1 is an essential factor in the acute response to proteotoxic stress, in which it causes rapid transcription of heat shock protein (HSP) genes in order to permit survival of cells and restoration of global protein quality. In addition to this property however, HSF1 is chronically activated or overexpressed in a wide range of cancers and is essential for multiple pathways of malignant transformation. Studies in recent years indicate a remarkable pleiotropy in the properties of HSF1 in cancer. HSF1 functions as a transcription factor for HSP genes, reminiscent of its role in the stress response, and the resultant elevation in HSP levels leads to a reduction in programmed cell death and senescence and permits overexpression of mutated oncogenic protein clients required to fuel tumor growth. In addition HSF1 plays a role as a signal modulator, stimulating kinase activity, regulating energy metabolism and permitting the development of polyploidy in cancer cells. HSF1 can also function as an inhibitor of transcription and in cooperation with NuRD family factors can repress genes that oppose metastasis. Inhibitors of HSF1 are undergoing selection and future studies may see the testing of HSF1 as a target in cancer therapy.

MO25 is a master regulator of SPAK/OSR1 and MST3/MST4/YSK1 protein kinases

Mouse protein-25 (MO25) isoforms bind to the STRAD pseudokinase and stabilise it in a conformation that can activate the LKB1 tumour suppressor kinase. We demonstrate that by binding to several STE20 family kinases, MO25 has roles beyond controlling LKB1. These new MO25 targets are SPAK/OSR1 kinases, regulators of ion homeostasis and blood pressure, and MST3/MST4/YSK1, involved in controlling development and morphogenesis. Our analyses suggest that MO25α and MO25β associate with these STE20 kinases in a similar manner to STRAD. MO25 isoforms induce approximately 100-fold activation of SPAK/OSR1 dramatically enhancing their ability to phosphorylate the ion cotransporters NKCC1, NKCC2 and NCC, leading to the identification of several new phosphorylation sites. siRNA-mediated reduction of expression of MO25 isoforms in mammalian cells inhibited phosphorylation of endogenous NKCC1 at residues phosphorylated by SPAK/OSR1, which is rescued by re-expression of MO25α. MO25α/β binding to MST3/MST4/YSK1 also stimulated kinase activity three- to four-fold. MO25 has evolved as a key regulator of a group of STE20 kinases and may represent an ancestral mechanism of regulating conformation of pseudokinases and activating catalytically competent protein kinases.

Structure of Lipid Kinase p110β/p85β Elucidates an Unusual SH2-Domain-Mediated Inhibitory Mechanism

SummaryPhosphoinositide 3-kinases (PI3Ks) are essential for cell growth, migration, and survival. The structure of a p110β/p85β complex identifies an inhibitory function for the C-terminal SH2 domain (cSH2) of the p85 regulatory subunit. Mutagenesis of a cSH2 contact residue activates downstream signaling in cells. This inhibitory contact ties up the C-terminal region of the p110β catalytic subunit, which is essential for lipid kinase activity. In vitro, p110β basal activity is tightly restrained by contacts with three p85 domains: the cSH2, nSH2, and iSH2. RTK phosphopeptides relieve inhibition by nSH2 and cSH2 using completely different mechanisms. The binding site for the RTK's pYXXM motif is exposed on the cSH2, requiring an extended RTK motif to reach and disrupt the inhibitory contact with p110β. This contrasts with the nSH2 where the pY-binding site itself forms the inhibitory contact. This establishes an unusual mechanism by which p85 SH2 domains contribute to RTK signaling specificities.

A role for the essential YycG sensor histidine kinase in sensing cell division

The YycG sensor histidine kinase co-ordinates cell wall remodelling with cell division in Gram-positive bacteria by controlling the transcription of genes for autolysins and their inhibitors. Bacillus subtilis YycG senses cell division and is enzymatically activated by associating with the divisome at the division septum. Here it is shown that the cytoplasmic PAS domain of this multi-domain transmembrane kinase is a determining factor translocating the kinase to the division septum. Furthermore, translocation to the division septum, per se, is insufficient to activate YycG, indicating that specific interactions and/or ligands produced there are required to stimulate kinase activity. N-terminal truncations of YycG lose negative regulation of their activity inferring that this regulation is accomplished through its transmembrane and extramembrane domains interacting with the membrane associated YycH and YycI proteins that do not localize to the divisome. The data indicate that YycG activity in non-dividing cells is suppressed by its interaction with YycH and YycI and its activation is co-ordinated to cell division in dividing cells by specific interactions that occur within the divisome.