Protein Tyr Kinases Research Articles

The intrinsic substrate specificity of the human tyrosine kinome

Phosphorylation of proteins on tyrosine (Tyr) residues evolved in metazoan organisms as a mechanism of coordinating tissue growth1. Multicellular eukaryotes typically have more than 50 distinct protein Tyr kinases that catalyse the phosphorylation of thousands of Tyr residues throughout the proteome1–3. How a given Tyr kinase can phosphorylate a specific subset of proteins at unique Tyr sites is only partially understood4–7. Here we used combinatorial peptide arrays to profile the substrate sequence specificity of all human Tyr kinases. Globally, the Tyr kinases demonstrate considerable diversity in optimal patterns of residues surrounding the site of phosphorylation, revealing the functional organization of the human Tyr kinome by substrate motif preference. Using this information, Tyr kinases that are most compatible with phosphorylating any Tyr site can be identified. Analysis of mass spectrometry phosphoproteomic datasets using this compendium of kinase specificities accurately identifies specific Tyr kinases that are dysregulated in cells after stimulation with growth factors, treatment with anti-cancer drugs or expression of oncogenic variants. Furthermore, the topology of known Tyr signalling networks naturally emerged from a comparison of the sequence specificities of the Tyr kinases and the SH2 phosphotyrosine (pTyr)-binding domains. Finally we show that the intrinsic substrate specificity of Tyr kinases has remained fundamentally unchanged from worms to humans, suggesting that the fidelity between Tyr kinases and their protein substrate sequences has been maintained across hundreds of millions of years of evolution.

Importance of Tyrosine Phosphorylation in Hormone-Regulated Plant Growth and Development.

Protein phosphorylation is the most frequent post-translational modification (PTM) that plays important regulatory roles in a wide range of biological processes. Phosphorylation mainly occurs on serine (Ser), threonine (Thr), and tyrosine (Tyr) residues, with the phosphorylated Tyr sites accounting for ~1–2% of all phosphorylated residues. Tyr phosphorylation was initially believed to be less common in plants compared to animals; however, recent investigation indicates otherwise. Although they lack typical protein Tyr kinases, plants possess many dual-specificity protein kinases that were implicated in diverse cellular processes by phosphorylating Ser, Thr, and Tyr residues. Analyses of sequenced plant genomes also identified protein Tyr phosphatases and dual-specificity protein phosphatases. Recent studies have revealed important regulatory roles of Tyr phosphorylation in many different aspects of plant growth and development and plant interactions with the environment. This short review summarizes studies that implicated the Tyr phosphorylation in biosynthesis and signaling of plant hormones.

β-Arrestin2 Is Critically Involved in the Differential Regulation of Phosphosignaling Pathways by Thyrotropin-Releasing Hormone and Taltirelin.

In recent years, thyrotropin-releasing hormone (TRH) and its analogs, including taltirelin (TAL), have demonstrated a range of effects on the central nervous system that represent potential therapeutic agents for the treatment of various neurological disorders, including neurodegenerative diseases. However, the molecular mechanisms of their actions remain poorly understood. In this study, we investigated phosphosignaling dynamics in pituitary GH1 cells affected by TRH and TAL and the putative role of β-arrestin2 in mediating these effects. Our results revealed widespread alterations in many phosphosignaling pathways involving signal transduction via small GTPases, MAP kinases, Ser/Thr- and Tyr-protein kinases, Wnt/β-catenin, and members of the Hippo pathway. The differential TRH- or TAL-induced phosphorylation of numerous proteins suggests that these ligands exhibit some degree of biased agonism at the TRH receptor. The different phosphorylation patterns induced by TRH or TAL in β-arrestin2-deficient cells suggest that the β-arrestin2 scaffold is a key factor determining phosphorylation events after TRH receptor activation. Our results suggest that compounds that modulate kinase and phosphatase activity can be considered as additional adjuvants to enhance the potential therapeutic value of TRH or TAL.

Human Sperm Capacitation Involves the Regulation of the Tyr-Phosphorylation Level of the Anion Exchanger 1 (AE1).

Bicarbonate uptake is one of the early steps of capacitation, but the identification of proteins regulating anion fluxes remains elusive. The aim of this study is to investigate the role of sperm solute carrier 4 (SLC4) A1 (spAE1) in the capacitation process. The expression, location, and tyrosine-phosphorylation (Tyr-P) level of spAE1 were assessed. Thereby, it was found that 4,4′-Diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS), an SLC4 family channel blocker, inhibited capacitation in a dose-dependent manner by decreasing acrosome reaction (ARC% 24.5 ± 3.3 vs. 64.9 ± 4.3, p < 0.05) and increasing the percentage of not viable cells (NVC%), comparable to the inhibition by I-172, a cystic fibrosis transmembrane conductance regulator (CFTR) blocker (AR% 30.5 ± 4.4 and NVC% 18.6 ± 2.2). When used in combination, a synergistic inhibitory effect was observed with a remarkable increase of the percentage of NVC (45.3 ± 4.1, p < 0.001). spAE1 was identified in sperm membrane as a substrate for Tyr-protein kinases Lyn and Syk, which were identified as both soluble and membrane-bound pools. spAE1-Tyr-P level increased in the apical region of sperm under capacitating conditions and was negatively affected by I-172 or DIDS, and, to a far greater extent, by a combination of both. In conclusion, we demonstrated that spAE1 is expressed in sperm membranes and it is phosphorylated by Syk, but above all by Lyn on Tyr359, which are involved in sperm viability and capacitation.

The multifunctional role of phospho-calmodulin in pathophysiological processes.

Calmodulin (CaM) is a versatile Ca2+-sensor/transducer protein that modulates hundreds of enzymes, channels, transport systems, transcription factors, adaptors and other structural proteins, controlling in this manner multiple cellular functions. In addition to its capacity to regulate target proteins in a Ca2+-dependent and Ca2+-independent manner, the posttranslational phosphorylation of CaM by diverse Ser/Thr- and Tyr-protein kinases has been recognized as an important additional manner to regulate this protein by fine-tuning its functionality. In this review, we shall cover developments done in recent years in which phospho-CaM has been implicated in signalling pathways that are relevant for the onset and progression of diverse pathophysiological processes. These include diverse systems playing a major role in carcinogenesis and tumour development, prion-induced encephalopathies and brain hypoxia, melatonin-regulated neuroendocrine disorders, hypertension, and heavy metal-induced cell toxicity.

Targeting Wnt signaling pseudokinases in hematological cancers.

Recent studies showed that several pseudokinases from the receptor tyrosine kinase family are important players in regulating cancer cell invasion, metastasis, and drug resistance, suggesting that targeting these proteins can play a therapeutic role in cancer treatment. Receptor Tyr kinase-like orphan receptors (RORs), protein Tyr kinase 7 (PTK7) (also called colon carcinoma kinase 4 (CCK4)), and receptor-like Tyr kinase (RYK) are Wnt ligand binding receptors within the non-canonical Wnt signaling, with important roles in development, tissue homeostasis, and organogenesis. At the cellular level, these receptors transduce signals important for cell survival, migration, polarization, and chemotaxis. Considerable progress has been made in the last decade in the field of pseudokinase signaling, improving our understanding of their structure-function mechanisms, and intracellular network of transduction components. Consequently, their role in various diseases, including cancer, is now scrutinized for therapeutic interventions to improve treatment outcome. In this article, we review findings regarding molecular mechanisms and targeted therapies for ROR1, PTK7, and RYK in hematological malignancies.

Constitutively Bound EGFR-Mediated Tyrosine Phosphorylation of TLR9 Is Required for Its Ability To Signal.

Mammalian TLRs recognize microbial infection or cell death-associated danger signals and trigger the appropriate cellular response. These responses determine the strength and the outcome of the host-microbe interaction. TLRs are transmembrane proteins located on the plasma or the endosomal membrane. Their ectodomains recognize specific microbial or endogenous ligands, and the cytoplasmic domains interact with specific proteins to activate intracellular signaling pathways. TLR9, an endosomal TLR, is activated by endocytosed DNA. Activated TLR9 recruits the cytoplasmic adapter MyD88 and other signaling proteins to induce the synthesis of inflammatory cytokines and IFN. Uncontrolled activation of TLR9 leads to the undesired overproduction of inflammatory cytokines and consequent pathogenesis. Therefore, appropriate activation and the regulation of TLR9 signaling are critical. Tyrosine (Tyr) phosphorylation of TLR9 is essential for its activation; however, the role of specific Tyr kinases is not clear. In this article, we report that epidermal growth factor receptor (EGFR), a membrane-bound protein Tyr kinase, is essential for TLR9 signaling. Genetic ablation of EGFR or pharmacological inhibition of its kinase activity attenuates TLR9-mediated induction of genes in myeloid and nonmyeloid cell types. EGFR is constitutively bound to TLR9; upon ligand stimulation, it mediates TLR9 Tyr phosphorylation, which leads to the recruitment of MyD88, activation of the signaling kinases and transcription factors, and gene induction. In mice, TLR9-mediated liver injury and death are blocked by an EGFR inhibitor or deletion of the EGFR gene from myeloid cells, which are the major producers of inflammatory cytokines.

Characterization of a eukaryotic-like protein kinase, DspB, with an atypical catalytic loop motif from Myxococcus xanthus

Serine (Ser)/threonine (Thr) or tyrosine (Tyr) protein kinases in eukaryotes contain RDxKxxN or RDx(A/R)A(A/R)N sequences, respectively, in the catalytic loop. Myxococcus xanthus DspB is a dual-specificity kinase that contains an atypical sequence, RDVAQKN, in the catalytic loop. The DspB mutant (A165K), which contains the canonical RDxKxxN motif, had an approximate 1.3-fold increase in kinase activity toward myelin basic protein (MBP). Arginine-aspartate (RD) kinases carry a conserved Arg immediately preceding the catalytic Asp that is required for autophosphorylation of the activation loop. DspB belongs to the RD kinase family and contains one Ser residue (Ser-190) and one Thr residue (Thr-194) in the activation loop. Mutation of Ser-190 or Thr-194 to Ala did not significantly affect the kinase activity toward MBP. We previously reported that four M. xanthus eukaryotic-like kinases (EPKs) are autophosphorylated on Tyr residues. These EPKs contain six Tyr residues at homologous positions, and five of those Tyr residues, Y25, Y102, Y145, Y173, and Y205, are conserved in DspB. DspB is mainly autophosphorylated on Y145, and a Y145F mutant has reduced kinase activity, suggesting that autophosphorylation of the Tyr residue of DspB may be required for high-level kinase activity.

Do plant chloroplasts contain histidine kinases?

Plastids are evolutionary descendants of cyanobacteria and retain many proteins of cyanobacterial origin including histidine kinases (HKs). Histidine kinases form a major group of protein kinases in cyanobacteria but a minor group in angiosperms; no HK has been detected in plant plastids so far. This raises the question: have higher plant plastids retained some cyanobacterial His/Asp regulatory systems or the latter have been replaced with Ser/Thr or Tyr protein kinases more typical for eukaryotes? To answer this question H1 antiserum against conservative phospho-acceptor motif of HKs was raised and applied for the analysis of different chloroplast fractions. In maize, three polypeptides with apparent molecular masses about 26, 27.5, and 28 kDa were revealed in thylakoid membranes. The results of in organello phosphorylation suggest that these proteins may be His-phosphorylated. Polypeptides with similar molecular masses were revealed in various mono- and dicotyledonous plants. Bioinformatic analysis demonstrated that in angiosperms these polypeptides might result from alternative transcription initiation and/or alternative processing of mRNAs of genes encoding well-known HKs. Besides, the genome of the moss Physcomitrella patens contains much more genes that could code for plastid HKs. The data obtained let us suppose that plastids of contemporary plants have HK(s) that could be involved in regulation of plastid gene expression.

Crystal Structures of Two Aminoglycoside Kinases Bound with a Eukaryotic Protein Kinase Inhibitor

Antibiotic resistance is recognized as a growing healthcare problem. To address this issue, one strategy is to thwart the causal mechanism using an adjuvant in partner with the antibiotic. Aminoglycosides are a class of clinically important antibiotics used for the treatment of serious infections. Their usefulness has been compromised predominantly due to drug inactivation by aminoglycoside-modifying enzymes, such as aminoglycoside phosphotransferases or kinases. These kinases are structurally homologous to eukaryotic Ser/Thr and Tyr protein kinases and it has been shown that some can be inhibited by select protein kinase inhibitors. The aminoglycoside kinase, APH(3′)-IIIa, can be inhibited by CKI-7, an ATP-competitive inhibitor for the casein kinase 1. We have determined that CKI-7 is also a moderate inhibitor for the atypical APH(9)-Ia. Here we present the crystal structures of CKI-7-bound APH(3′)-IIIa and APH(9)-Ia, the first structures of a eukaryotic protein kinase inhibitor in complex with bacterial kinases. CKI-7 binds to the nucleotide-binding pocket of the enzymes and its binding alters the conformation of the nucleotide-binding loop, the segment homologous to the glycine-rich loop in eurkaryotic protein kinases. Comparison of these structures with the CKI-7-bound casein kinase 1 reveals features in the binding pockets that are distinct in the bacterial kinases and could be exploited for the design of a bacterial kinase specific inhibitor. Our results provide evidence that an inhibitor for a subset of APHs can be developed in order to curtail resistance to aminoglycosides.

Abstract 1636: ErbB4 is a new therapeutic target for metastasis and anoikis resistance in Ewing's sarcoma

Abstract Most Ewing sarcoma (ES)-related deaths occur as a result of metastatic disease. As a barrier to metastases, cells normally undergo a form of cell death called ‘‘anoikis” after they lose contact with the extracellular matrix or neighboring cells. Tumor cells that acquire malignant potential develop mechanisms to suppress anoikis. We previously demonstrated that ES tumors suppress anoikis and show prolonged survival as multicellular spheroids, which mimic micrometastases under anchorage-independent conditions. Here we have used the paired CHLA-9 primary tumor line established at diagnosis and the metastatic CHLA-10 line established from the same patient after four cycles of induction chemotherapy with cisplatin, doxorubicin, cyclophosphamide, and etoposide, as well as high-throughput Affymetrix whole exon array platforms to screen for potential therapeutic targets to treat metastatic disease. We have identified ErbB4, a member of the epidermal growth factor receptor (EGFR) family, as a novel mediator of anoikis resistance in ES. We detected a 4-fold increase in ERBB4, along with enhanced ErbB4 protein expression and Tyr kinase activation (confirmed by P-Tyr kinase proteome arrays) in CHLA-10 cells grown under anchorage independent conditions, compared to CHLA-9 tumor cells under the same conditions. Upregulated ErbB4 expression was further confirmed in a panel of metastatic vs. primary ES cell lines at both the mRNA and protein levels. We are currently validating these findings in the clinical setting by analyzing ErbB4 expression in paired primary vs. metastatic biopsies from ES patients. shRNA-mediated knockdown of ErbB4, or treatment with the ErbB inhibitor Lapatinib, dramatically reduced proliferation, decelerated tumor invasion, and sensitized metastatic/chemoresistant ES tumors to chemotherapeutic drugs used to treat ES patients. Taken together, our findings identify ErbB4 as a novel target for metastasis and anoikis resistance in ES. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1636. doi:10.1158/1538-7445.AM2011-1636

Diacylglycerol-Stimulated Endocytosis of Transferrin in Trypanosomatids Is Dependent on Tyrosine Kinase Activity

Small molecule regulation of cell function is an understudied area of trypanosomatid biology. In Trypanosoma brucei diacylglycerol (DAG) stimulates endocytosis of transferrin (Tf). However, it is not known whether other trypanosomatidae respond similarly to the lipid. Further, the biochemical pathways involved in DAG signaling to the endocytic system in T. brucei are unknown, as the parasite genome does not encode canonical DAG receptors (e.g. C1-domains). We established that DAG stimulates endocytosis of Tf in Leishmania major, and we evaluated possible effector enzymes in the pathway with multiple approaches. First, a heterologously expressed glycosylphosphatidylinositol phospholipase C (GPI-PLC) activated endocytosis of Tf 300% in L. major. Second, exogenous phorbol ester and DAGs promoted Tf endocytosis in L. major. In search of possible effectors of DAG signaling, we discovered a novel C1-like domain (i.e. C1_5) in trypanosomatids, and we identified protein Tyr kinases (PTKs) linked with C1_5 domains in T. brucei, T. cruzi, and L. major. Consequently, we hypothesized that trypanosome PTKs might be effector enzymes for DAG signaling. General uptake of Tf was reduced by inhibitors of either Ser/Thr or Tyr kinases. However, DAG-stimulated endocytosis of Tf was blocked only by an inhibitor of PTKs, in both T. brucei and L. major. We conclude that (i) DAG activates Tf endocytosis in L. major, and that (ii) PTKs are effectors of DAG-stimulated endocytosis of Tf in trypanosomatids. DAG-stimulated endocytosis of Tf may be a T. brucei adaptation to compete effectively with host cells for vertebrate Tf in blood, since DAG does not enhance endocytosis of Tf in human cells.

Protein tyrosine kinases and protein tyrosine phosphatases are involved in abscisic acid-dependent processes in Arabidopsis seeds and suspension cells.

Protein tyrosine (Tyr) phosphorylation plays a central role in many signaling pathways leading to cell growth and differentiation in animals. Tyr phosphorylated proteins have been detected in higher plants, and the roles of protein Tyr phosphatases and protein Tyr kinases in some physiological responses have been shown. We investigated the involvement of Tyr phosphorylation events in abscisic acid (ABA) signaling using a pharmacological approach. Phenylarsine oxide, a specific inhibitor of protein Tyr phosphatase activity, abolished the ABA-dependent accumulation of RAB18 (responsive to ABA 18) transcripts. Protein Tyr kinase inhibitors like genistein, tyrphostin A23, and erbstatin blocked the RAB18 expression induced by ABA in Arabidopsis (Arabidopsis thaliana). Stomatal closure induced by ABA was also inhibited by phenylarsine oxide and genistein. We studied the changes in the Tyr phosphorylation levels of proteins in Arabidopsis seeds after ABA treatment. Proteins were separated by two-dimensional gel electrophoresis, and those phosphorylated on Tyr residues were detected using an anti-phosphotyrosine antibody by western blot. Changes were detected in the Tyr phosphorylation levels of 19 proteins after ABA treatment. Genistein inhibited the ABA-dependent Tyr phosphorylation of proteins. The 19 proteins were analyzed by matrix-assisted laser-desorption ionization time-of-flight/time-of-flight mass spectrometry. Among the proteins identified were storage proteins like cruciferins, enzymes involved in the mobilization of lipid reserves like aconitase, enolase, aldolase, and a lipoprotein, and enzymes necessary for seedling development like the large subunit of Rubisco. Additionally, the identification of three putative signaling proteins, a peptidyl-prolyl isomerase, an RNA-binding protein, and a small ubiquitin-like modifier-conjugating enzyme, enlightens how Tyr phosphorylation might regulate ABA transduction pathways in plants.

PETPhos: A customized expression vector designed for further characterization of Ser/Thr/Tyr protein kinases and their substrates

Bacterial genomics revealed the widespread distribution of serine/threonine protein kinases (STPKs), which regulate various cellular processes. However, understanding the role of phosphorylation in prokaryotes has been hampered by the paucity of endogenous substrates identified and the restricted number of tools allowing identification and characterization of the phosphoresidues. Herein, we describe an improved vector, pETPhos, to express proteins harboring a N-terminal His-tag fusion, which can be efficiently removed using the TEV protease. One major advantage of pETPhos relies on the lack of Ser and Thr residues in the fusion tag, representing potential non-specific phosphorylation sites. The usefulness of pETPhos is illustrated by a comparative analysis in which the Mycobacterium tuberculosis protein Rv2175c, a substrate of the STPK PknL, is expressed either in a pET28 derivative or in pETPhos. Following in vitro phosphorylation with PknL, phosphoaminoacid analysis revealed the presence of phosphorylated Ser and Thr in Rv2175c expressed in the pET28 derivative. However, when expressed in pETPhos, only Thr were phosphorylated. These findings indicate that STPKs can phosphorylate Ser-containing His-tag fusions, thus conducting to misleading results. We demonstrate that pETPhos represents a valuable tool for characterization of the phosphoacceptors in bacterial STPKs, and presumably also in Tyr protein kinases, as well as in their substrates.

Growth and survival signaling by lipid kinases

The phosphoinositide 3‐kinase (PI3K) pathway plays a critical role in cell growth regulation. PI3K generates the lipid, phosphatidylinositol‐3,4,5‐trisphosphate (PIP3), that acts as a membrane bound second messenger to activate AKT/PKB family protein Ser/Thr kinases as well Tec family protein‐Tyr kinases. PIP3 also regulates proteins that control Arf, Rac and Ras family GTP‐binding proteins. This lipid is elevated in cancers, either due to loss of PTEN, the phosphatases that degrades it, or due to constitutive PI3K activity because of activating mutations in PIK3CA or an upstream activator of PI3K. The ultimate consequence of activating PI3K is to generate changes in signaling networks and gene expression patterns that promote cell growth, cell survival and cell movement. In order to elucidate the role of the PI3K pathway in normal cell growth and in cancer, we have generated mice in which genes for PI3K are deleted or activated in specific tissues. In addition, we have investigated the biochemical mechanisms by which PI3K becomes activated in human cancers. Our progress in these areas will be summarized.

Phosphoinositide 3–kinase γ participates in T cell receptor–induced T cell activation

Class I phosphoinositide 3–kinases (PI3Ks) constitute a family of enzymes that generates 3-phosphorylated polyphosphoinositides at the cell membrane after stimulation of protein tyrosine (Tyr) kinase–associated receptors or G protein–coupled receptors (GPCRs). The class I PI3Ks are divided into two types: class IA p85/p110 heterodimers, which are activated by Tyr kinases, and the class IB p110γ isoform, which is activated by GPCR. Although the T cell receptor (TCR) is a protein Tyr kinase–associated receptor, p110γ deletion affects TCR-induced T cell stimulation. We examined whether the TCR activates p110γ, as well as the consequences of interfering with p110γ expression or function for T cell activation. We found that after TCR ligation, p110γ interacts with Gαq/11, lymphocyte-specific Tyr kinase, and ζ-associated protein. TCR stimulation activates p110γ, which affects 3-phosphorylated polyphosphoinositide levels at the immunological synapse. We show that TCR-stimulated p110γ controls RAS-related C3 botulinum substrate 1 activity, F-actin polarization, and the interaction between T cells and antigen-presenting cells, illustrating a crucial role for p110γ in TCR-induced T cell activation.

SHP‐1 Tyrosine Phosphatase in Human Erythrocytes

SHP-1 is a SH2-domain containing protein Tyr-phosphatase expressed in hematopoietic cell lines, which is hypothesized to play a negative role in signal transduction. In human erythrocytes, the phospho-Tyr level of proteins, mainly transmembrane band 3, is closely controlled by the antithetic activity of Tyr-protein kinases and phosphatases, resulting in a dephosphorylated state. Only after particular stimuli, as with oxidizing agents, diamide or pervanadate, or thiol alkylating compound, N-ethyl maleimide (NEM), Tyr-phosphorylation of band 3 can be triggered, inhibiting Tyr-phosphatase action and inducing erythrocyte membrane reorganization. We demonstrate that, in human erythrocytes, SHP-1 is present in membranes from resting cells, but in 5% of the protein amount. Interestingly, this amount increases up to threefold following NEM treatment of intact cells, whereas diamide and pervanadate do not alter the normal protein location. In addition, SHP-1 translocation from cytosol to membrane is not affected by band 3 P-Tyr level, because it is not mediated by the SH2-P-Tyr recruitment mechanism, and localizes into the cytoskeletal compartment. Band 3 is the target of SHP-1, which dephosphorylates Tyr 8, 21, and 904. These findings support the idea that, in human erythrocytes, the normal level of Tyr-phosphorylation of membrane protein, mainly band 3, must be downregulated. We hypothesize that the presence of both SHP-2 and SHP-1 ensures band 3 dephosphorylation in different conditions: SHP-2, through interaction of its SH2 domain/s to P-Tyr protein, is regulated by the band 3 Tyr-phosphorylation level; SHP-1 may be involved by simple membrane rearrangement.

Dusty protein kinases: Primary structure, gene evolution, tissue specific expression and unique features of the catalytic domain

Ser/Thr- and Tyr-Protein kinases constitute a key switch underlying the dynamic nature and graded regulation of signal transduction and pathway activities in cellular organization. Here we describe the identification and characterization of Dusty, a single-copy gene that arose in metazoan evolution and encodes a putative dual Ser/Thr and Tyr protein kinase with unique structural features. Dusty is widely expressed in vertebrates, broadly distributed in the central nervous system, and deregulated in certain human cancers. Confocal imaging of transiently expressed human Dusty-GFP fusion proteins showed a cytoplasmic distribution. Dusty proteins from lower to higher species display an increasing degree of sequence conservation from the N-terminal non-catalytic domain to C-terminal catalytic domain. The non-catalytic region has eight conserved cysteine residues, multiple potential kinase-docking motifs and phosphorylation sites, whereas the catalytic domain is divergent and about equally distant of Ser/Thr and Tyr protein kinases. Homology analyses identified the essential catalytic residues, suggesting that Dusty homologues all possess the enzymatic activity of a protein kinase. Taken together, Dusty is a unique evolutionarily selected group of divergent protein kinases that may play important functional roles in the brain and other tissues of vertebrates.

Peptide Microarray Analysis of Substrate Specificity of the Transmembrane Ser/Thr Kinase KPI-2 Reveals Reactivity with Cystic Fibrosis Transmembrane Conductance Regulator and Phosphorylase

Human lemur (Lmr) kinases are predicted to be Tyr kinases based on sequences and are related to neurotrophin receptor Trk kinases. This study used homogeneous recombinant KPI-2 (Lmr2, LMTK2, Cprk, brain-enriched protein kinase) kinase domain and a library of 1,154 peptides on a microarray to analyze substrate specificity. We found that KPI-2 is strictly a Ser/Thr kinase that reacts with Ser either preceded by or followed by Pro residues but unlike other Pro-directed kinases does not strictly require an adjacent Pro residue. The most reactive peptide in the library corresponds to Ser-737 of cystic fibrosis transmembrane conductance regulator, and the recombinant R domain of cystic fibrosis transmembrane conductance regulator was a preferred substrate. Furthermore the KPI-2 kinase phosphorylated peptides corresponding to the single site in phosphorylase and purified phosphorylase b, making this only the second known phosphorylase b kinase. Phosphorylase was used as a specific substrate to show that KPI-2 is inhibited in living cells by addition of nerve growth factor or serum. The results demonstrate the utility of the peptide library to probe specificity and discover kinase substrates and offer a specific assay that reveals hormonal regulation of the activity of this unusual transmembrane kinase.

Dusty Protein Kinase in Vertebrates: Tissue‐specific Expression, Gene Evolution and Unique Structural Features

Protein kinases catalyze phosphate transfer from ATP to the Ser/Thr- or Tyr-OH groups of protein substrates and have undergone a productive expansion as a family from prokaryotes to metazoans. Here we characterized Dusty, a putative dual Ser/Thr and Tyr protein kinase with potential catalytic activity and unique structural properties. Dusty is a conserved single-copy gene present in all vertebrates and is expressed in multiple tissues, particularly broadly in the central nervous system of human, rat, mouse and chicken. Confocal imaging of Dusty-GFP fusion proteins localized Dusty to the cytoplasm with a punctate distribution. Dusty proteins show increased sequence conservation from the N-terminal non-kinase part to C-terminal catalytic domain. The non-kinase part has no homology with any known conserved domains but multiple potential kinase-docking motifs and phosphorylation sites. The catalytic domain is composite in nature whose N and C-termini bear features conserved in Ser/Thr and Tyr kinase domains, respectively. Comparative analysis of Dusty kinase domains identified the conserved pattern of catalytically essential residues, and structure modeling suggests a catalytic activity. Based on these results, we conclude that Dusty is not a RIP family member, but a unique hybrid of Ser/Thr and Tyr kinases that survived natural selection and may possess novel functional roles in the brain and other tissues.