Critical Proline Residues Research Articles

"Cyclophilin A" Enzymatic Effect on the Aggregation Behavior of 1N4R Tau Protein: An Overlooked Crucial Determinant that should be Re-considered in Alzheimer's Disease Pathogenesis.

Neurodegenerative disorders like Alzheimer's disease (AD) involve the abnormal aggregation of tau protein, which forms toxic oligomers and amyloid deposits. The structure of tau protein is influenced by the conformational states of distinct proline residues, which are regulated by peptidyl-prolyl isomerases (PPIases). However, there has been no research on the impact of human cyclophilin A (CypA) as a PPIase on (non-phosphorylated) tau protein aggregation. On the basis of these explanations, we used various spectroscopic techniques to explore the effects of CypA on tau protein aggregation behavior. We demonstrated the role of the isomerization activity of CypA in promoting the formation of tau protein amyloid fibrils with well-defined and highly ordered cross-β structures. According to the "cistauosis hypothesis," CypA's ability to enhance tau protein fibril formation in AD is attributed to the isomerization of specific proline residues from the trans to cis configuration. To corroborate this theory, we conducted refolding experiments using lysozyme as a model protein. The presence of CypA increased lysozyme aggregation and impeded its refolding process. It is known that proper refolding of lysozyme relies on the correct (trans) isomerization of two critical proline residues. Thus, our findings confirmed that CypA induces the trans-to-cis isomerization of specific proline residues, ultimately leading to increased aggregation. Overall, this study highlights the emerging role of isomerization in tau protein pathogenesis in AD.

Encoding the β-Arrestin Trafficking Fate of Ghrelin Receptor GHSR1a: C-Tail-Independent Molecular Determinants in GPCRs.

G-protein-coupled receptors (GPCRs) can bias signaling through distinct biochemical pathways that originate from G-protein/receptor and β-arrestin/receptor complexes. Receptor conformations supporting β-arrestin engagement depend on multiple receptor determinants. Using ghrelin receptor GHR1a, we demonstrate by bioluminescence resonance energy transfer and fluorescence microscopy a critical role for its second intracellular loop 2 (ICL2) domain in stabilizing β-arrestin/GHSR1a core interactions and determining receptor trafficking fate. We validate our findings in ICL2 gain- and loss-of-function experiments assessing β-arrestin and ubiquitin-dependent internalization of the CC chemokine receptor, CCR1. Like all CC and CXC subfamily chemokine receptors, CCR1 lacks a critical proline residue found in the ICL2 consensus domain of rhodopsin-family GPCRs. Our study indicates that ICL2, C-tail determinants, and the orthosteric binding pocket that regulates β-arrestin/receptor complex stability are sufficient to encode a broad repertoire of the trafficking fates observed for rhodopsin-family GPCRs, suggesting they provide the essential elements for regulating a large fraction of β-arrestin signaling bias.

Ate1 Controls Cellular Warburg Effects by Modifying Hif1a with Arginylation

The hypoxia‐inducible factor 1a (HIF1a) is a key regulator of stress response and metabolism. Its expression level is known to be controlled by oxygen sensing with two critical proline residues followed by ubiquitination via the pVHL pathway. Here, we report that the degradation of HIF1a is also controlled by N‐terminal arginylation mediated by Arginyltransferase 1 (Ate1) according to the N‐end rule pathway. We found that this mechanism is separate from the classical pVHL‐pathway and it affects cellular Warburg effect, which is the preferred usage of glycolysis that are commonly seen in cancer cells. Furthermore, we found that Ate1, the only arginylation enzyme in animals, appears to have originated from mitochondrial gene transfer. At least a subset of Ate1 is located inside mitochondria and appears to be essential for the composition and functions of respiration complex II. Our study therefore suggest Ate1 as a master regulator of metabolism in cancer cells through arginylation modification of HIF1a.Support or Funding InformationThis study is supported by NIGMS R01 GM 107333, and a start‐up funding from the University of Miami.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Critical residues involved in tau binding to fyn: implications for tau phosphorylation in Alzheimer's disease.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterised by neuropathological deposits of amyloid plaques and neurofibrillary tangles comprised of β-amyloid and tau protein, respectively. In AD, tau becomes abnormally phosphorylated and aggregates to form intracellular deposits. However, the mechanisms by which tau exerts neurotoxicity in disease remain unclear. Recent studies have suggested that the presence of tau at synapses may indicate a role in neuronal signalling, which could be disrupted in pathological conditions. The non-receptor-associated tyrosine kinase fyn is located at the dendrite in neurons, where it was recently shown to interact with tau to stabilise receptor complexes at the post-synaptic density. Fyn also co-localises with tau in a proportion of neurons containing tau tangles in AD and fyn is also a tau kinase. Hence, tau-fyn interactions could play a pathogenic role in AD. Here we report the identification of critical proline residues, Pro213, Pro216, and Pro219, located within the fifth and sixth Pro-X-X-Pro motifs in the proline-rich region of tau, that are important for its binding to fyn. These residues in tau are flanked by numerous phosphorylation sites and therefore we investigated the relationship between fyn and the degree of tau phosphorylation in human post-mortem brain tissue. We found no difference in the amount of fyn present in control and AD brain. Notably, however, there was a significant correlation between fyn and phosphorylated tau at specific phospho-epitopes in control, but not in AD brain. Our results suggest that the pathological mechanisms underlying AD, that result in increased tau phosphorylation, may disrupt the physiological relationship between tau phosphorylation and fyn.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-016-0317-4) contains supplementary material, which is available to authorized users.

Hydroxylation-Dependent Interaction of Substrates to the Von Hippel-Lindau Tumor Suppressor Protein (VHL).

Oxygen-dependent hydroxylation of critical proline residues, catalyzed by prolyl hydroxylase (PHD1-3) enzymes, is a crucial posttranslational modification (PTM) within the canonical hypoxia-inducible factor (HIF)-centric cellular oxygen-sensing pathway. Alteration of substrates in this way often leads to proteasomal degradation mediated by the von Hippel-Lindau Tumor Suppressor protein (VHL) containing E3-ubiquitin ligase complex known as ECV (Elongins B/C, CUL2, VHL). Here, we outline in vitro protocols to demonstrate the ability of VHL to bind to a prolyl-hydroxylated substrate.

Proline-15 creates an amphipathic wedge in maculatin 1.1 peptides that drives lipid membrane disruption

The membrane interaction of peptides derived from maculatin 1.1 and caerin 1.1, with the sequence motif of N and C termini of maculatin 1.1, was compared in order to understand the role of these common sequence motifs, which encompass critical proline residues, on peptide secondary structure and on membrane binding and disruption in zwitterionic and anionic membranes. The peptides incorporated a single substitution with lysine or deletion of the central region to mimic the length of the antimicrobial peptides, citropin 1.1 and aurein 1.2. The impact of these changes in the sequence, length and physicochemical properties, on lytic activity and structure was assessed by dye-release from lipid vesicles and the change in the bilayer order as a function of membrane-bound peptide mass. All peptides adopted similar degrees of helical structure in both membrane systems. In addition, all peptide analogues were less active than either maculatin 1.1 or caerin 1.1 in dye release assays. The membrane binding was analyzed by dual polarization interferometry and the results showed that membrane binding was significantly affected by changes in the hydrophobic environment of Pro-15. Moreover, changes in the relative distribution of charge and hydrophobicity flanking Pro-15 also caused significant changes to the membrane order. Overall, the proline residue plays an important role in inducing a peptide structure that enhances the activity of these antimicrobial peptides.

Pseudosubstrate Regulation of the Type II P21-Activated Kinases (PAKs)

The type II p21-activated kinases (PAKs) are key effectors of RHO-family GTPases involved in cell motility, survival and proliferation. There are two sub-groups of PAK kinase, denoted type I (PAK1, PAK2 and PAK3) and type II (PAK4, PAK5 and PAK6). PAK4 is the best-studied type II PAK family member, is widely expressed, and is essential for viability in mice. PAK4 is required for oncogenic or metastatic phenotypes of many cancer cell lines, can promote tumorigenesis and breast cancer cell migration, and is the only PAK that is transforming when overexpressed. Furthermore, a specific PAK4 inhibitor (PF-3758309) has shown efficacy in mouse models of cancer. Using a structure-guided approach, we discovered that type II PAKs are regulated by an N-terminal autoinhibitory pseudosubstrate motif centered on a critical proline residue, and that this regulation occurs independently of activation loop phosphorylation. We determined 6 X-ray crystal structures of either full-length PAK4 or its catalytic domain, that demonstrate the molecular basis for pseudosubstrate binding to the active state with phosphorylated activation loop. We show that full-length PAK4 is constitutively autoinhibited, but mutation of the pseudosubstrate releases this inhibition and causes increased phosphorylation of BAD and cellular morphological changes. We also find that PAK6 is regulated by the pseudosubstrate region, indicating a common type II PAK autoregulatory mechanism. Finally, we find Src SH3, but not β-PIX SH3 can activate PAK4. This clear switch-like ‘on-off’ regulation mechanism for the type II PAKs is distinct from regulation mechanisms previously observed for the type I PAKs. We therefore provide new understanding for type II PAK regulation.

Type II p21-activated kinases (PAKs) are regulated by an autoinhibitory pseudosubstrate

The type II p21-activated kinases (PAKs) are key effectors of RHO-family GTPases involved in cell motility, survival, and proliferation. Using a structure-guided approach, we discovered that type II PAKs are regulated by an N-terminal autoinhibitory pseudosubstrate motif centered on a critical proline residue, and that this regulation occurs independently of activation loop phosphorylation. We determined six X-ray crystal structures of either full-length PAK4 or its catalytic domain, that demonstrate the molecular basis for pseudosubstrate binding to the active state with phosphorylated activation loop. We show that full-length PAK4 is constitutively autoinhibited, but mutation of the pseudosubstrate releases this inhibition and causes increased phosphorylation of the apoptotic regulation protein Bcl-2/Bcl-X(L) antagonist causing cell death and cellular morphological changes. We also find that PAK6 is regulated by the pseudosubstrate region, indicating a common type II PAK autoregulatory mechanism. Finally, we find Src SH3, but not β-PIX SH3, can activate PAK4. We provide a unique understanding for type II PAK regulation.

The Steady-State Repertoire of Human SCF Ubiquitin Ligase Complexes Does Not Require Ongoing Nedd8 Conjugation

The human genome encodes 69 different F-box proteins (FBPs), each of which can potentially assemble with Skp1-Cul1-RING to serve as the substrate specificity subunit of an SCF ubiquitin ligase complex. SCF activity is switched on by conjugation of the ubiquitin-like protein Nedd8 to Cul1. Cycles of Nedd8 conjugation and deconjugation acting in conjunction with the Cul1-sequestering factor Cand1 are thought to control dynamic cycles of SCF assembly and disassembly, which would enable a dynamic equilibrium between the Cul1-RING catalytic core of SCF and the cellular repertoire of FBPs. To test this hypothesis, we determined the cellular composition of SCF complexes and evaluated the impact of Nedd8 conjugation on this steady-state. At least 42 FBPs assembled with Cul1 in HEK 293 cells, and the levels of Cul1-bound FBPs varied by over two orders of magnitude. Unexpectedly, quantitative mass spectrometry revealed that blockade of Nedd8 conjugation led to a modest increase, rather than a decrease, in the overall level of most SCF complexes. We suggest that multiple mechanisms including FBP dissociation and turnover cooperate to maintain the cellular pool of SCF ubiquitin ligases.

Hearts of Hypoxia-inducible Factor Prolyl 4-Hydroxylase-2 Hypomorphic Mice Show Protection against Acute Ischemia-Reperfusion Injury

Hypoxia-inducible factor (HIF) has a pivotal role in oxygen homeostasis and cardioprotection mediated by ischemic preconditioning. Its stability is regulated by HIF prolyl 4-hydroxylases (HIF-P4Hs), the inhibition of which is regarded as a promising strategy for treating diseases such as anemia and ischemia. We generated a viable Hif-p4h-2 hypomorph mouse line (Hif-p4h-2(gt/gt)) that expresses decreased amounts of wild-type Hif-p4h-2 mRNA: 8% in the heart; 15% in the skeletal muscle; 34-47% in the kidney, spleen, lung, and bladder; 60% in the brain; and 85% in the liver. These mice have no polycythemia and show no signs of the dilated cardiomyopathy or hyperactive angiogenesis observed in mice with broad spectrum conditional Hif-p4h-2 inactivation. We focused here on the effects of chronic Hif-p4h-2 deficiency in the heart. Hif-1 and Hif-2 were stabilized, and the mRNA levels of glucose transporter-1, several enzymes of glycolysis, pyruvate dehydrogenase kinase 1, angiopoietin-2, and adrenomedullin were increased in the Hif-p4h-2(gt/gt) hearts. When isolated Hif-p4h-2(gt/gt) hearts were subjected to ischemia-reperfusion, the recovery of mechanical function and coronary flow rate was significantly better than in wild type, while cumulative release of lactate dehydrogenase reflecting the infarct size was reduced. The preischemic amount of lactate was increased, and the ischemic versus preischemic [CrP]/[Cr] and [ATP] remained at higher levels in Hif-p4h-2(gt/gt) hearts, indicating enhanced glycolysis and an improved cellular energy state. Our data suggest that chronic stabilization of Hif-1alpha and Hif-2alpha by genetic knockdown of Hif-p4h-2 promotes cardioprotection by induction of many genes involved in glucose metabolism, cardiac function, and blood pressure.

The effect of a proline residue on the rate of growth and the space group of α-spectrin SH3-domain crystals

alpha-Spectrin SH3-domain (Spc-SH3) crystallization is characterized by very fast growth of the crystals in the presence of ammonium sulfate as a precipitant agent. The origin of this behaviour can be attributed to the presence of a proline residue that participates in a crystal contact mimicking the binding of proline-rich sequences to SH3 domains. This residue, Pro20, is located in the RT loop and is the main contact in one of the interfaces present in the orthorhombic Spc-SH3 crystal structures. In order to understand the molecular interactions that are responsible for the very fast crystal growth of the wild-type (WT) Spc-SH3 crystals, the crystal structure of a triple mutant in which the residues Ser19-Pro20-Arg21 in the RT loop have been replaced by Gly19-Asp20-Ser21 (GDS Spc-SH3 mutant) has been solved. The removal of the critical proline residue results in slower nucleation of the Spc-SH3 crystals and a different arrangement of the protein molecules in the unit cell, leading to a crystal that belongs to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = b = 42.231, c = 93.655 A, and that diffracts to 1.45 A resolution. For both WT Spc-SH3 and the GDS mutant, light-scattering experiments showed that a dimer was formed in solution within a few minutes of the addition of 2 M ammonium sulfate at pH 6.5 and allowed the proposal of a mechanism for the nucleation and crystal growth of Spc-SH3 in which the Pro20 residue plays a key role in the rate of crystal growth.

HIF‐1 specific prolyl hydroxylases in elite athletes

The hypoxia sensitive transcription factor HIF‐1 has been suggested to be involved in skeletal muscle adaptation to endurance‐type exercise training. HIF‐1 protein levels are regulated by hydroxylation of critical proline residues and subsequent protein degradation of the alpha subunit. Three prolyl hydroxylation domain‐containing proteins (PHD1‐3) have been identified in mammalian cells, PHD2 appears to be the predominant form that regulates HIF‐1alpha protein stability in vivo1. HIF‐1 is stabilised in response to an acute bout of exercise in sedentary individuals2 but there is debate regarding the activity of the HIF‐1 system in response to long term training. Skeletal muscle biopsies were obtained from the vastus lateralis muscle at rest from 12 males with high oxidative capacity and 9 normally trained male controls. PHD2 mRNA displayed the highest expression levels of the three forms in human skeletal muscle, regardless of training status. Furthermore, PHD2 and 3 mRNA levels were both higher in elite athletes compared to the control individuals and PHD2 protein levels were significantly higher in the elite group. Conclusion: The HIF‐1 regulating prolyl hydroxylase PHD2 was significantly higher in elite athletes, which may explain an attenuated HIF‐1 activity in response to long term endurance training. This work was supported by the Swedish Research Council, the Swedish National Centre for Research in Sports

Single Proline Residues can Dictate the Oxidative Folding Pathways of Cysteine-rich Peptides

The cysteine-rich N and C-terminal domains of minicollagen-1 from Hydra nematocysts fold with excesses of oxidized/reduced glutathione (10:1) into globular structures with distinct cystine frameworks despite their identical cysteine sequence pattern. An additional main difference is the cis conformation of a conserved proline residue in the N-terminal and the trans conformation of this residue in the C-terminal domain. Comparative analysis of the oxidative folding revealed for the C-terminal domain a fast and highly cooperative formation of a single disulfide isomer. Conversely, oxidation of the N-terminal domain proceeds mainly via an intermediate that results from the fast quasi-stochastic disulfide formation according to the proximity rule. The rate of conversion of the bead-like isomer into the globular end-product is largely dominated by the trans-to-cis isomerization of the critical proline residue as well assessed by its replacement with (4R)- and (4S)-fluoroproline known to exhibit distinct propensities for the trans and cis conformation, respectively. Independently, whether the trans or cis conformation is favored by these substitutions, both analogues retain sufficient sequence-encoded information to fold almost quantitatively into the identical cystine framework and thus spatial structure of the parent peptide with the critical proline residue as cis isomer, but at rates significantly lower for the (4R) than for the (4S)-fluoroproline analogue. Correspondingly, other sequence-encoded structural elements have to act as a driving force for these unidirectional folding pathways despite the rather simple sequence composition consisting only of aliphatic residues, some proline and only one aromatic residue (tyrosine) in the core parts of the C and N-terminal domains. The two cysteine-rich domains of minicollagen-1 may well represent ideal targets for ab initio structure calculations in order to learn more about the elementary information encoded in such primordial molecules.

Antitumor effects of new proline analogs on cancer cell lines in vitro

3164 Background: Components of the extracellular matrix (ECM), especially collagens, are involved in tumor cell development, proliferation and dissemination and therefore represent a potential target for tumor therapy. The ECM-associated proline-rich structures are susceptible to suitable toxic proline analogs, which disrupt protein assembly and functions. Methods: An extensive panel of proline analogs (lead compound: cis-4-hydroxy-L-proline; CHP) and the collagen inhibitor L-azetidine-2-carboxylic acid were tested for cytotoxicity against a large panel of cell lines. In addition to a tetrazolium-based assay (MTT), cell cycle analysis (propidium iodide) and apoptosis assays (annexin V) were performed. Amino acid uptake was studied using radioactive proline and differentiation was assessed by measuring induction of alkaline phosphatase (ALP). Results: Antiproliferative activity of CHP was found in cell lines derived from colon carcinomas, pancreatic, prostatic, and breast cancer as well as osteosarcoma and glioma (IC50: 35 - 175 μg/ml) and increased activity was detected under acidic extracellular conditions prevailing in solid tumors. Cell cycle arrest was found preferentially in G2M and apoptotic cells were detectable following prolonged exposure (7 - 10 days). CHP treatment of 2 colon carcinoma cell lines (HT29, CaCo-2) resulted in induction of the differentiation marker ALP. Proline competition tests indicated uptake of CHP via alpha-(methylamino)-isobutyric acid (MeAIB)-sensitive amino acid transport. Extensive testing of other cytotoxic proline-related compounds led to the characterization of N-methylated CHP derivatives, which show no interference with transport of proline, and proved to be capable of enhancing the CHP-induced toxicity by up to 30 %. Conclusions: Highly efficient proline analogs in combination exhibit considerable toxicity in vitro against diverse cell lines and should be studied in extended clinical CHP trials as agents modulating tumor cell growth by targeting ECM and/or critical proline residues of tumor-associated proteins. No significant financial relationships to disclose.

Antitumor effects of new proline analogs on cancer cell lines in vitro

3164 Background: Components of the extracellular matrix (ECM), especially collagens, are involved in tumor cell development, proliferation and dissemination and therefore represent a potential target for tumor therapy. The ECM-associated proline-rich structures are susceptible to suitable toxic proline analogs, which disrupt protein assembly and functions. Methods: An extensive panel of proline analogs (lead compound: cis-4-hydroxy-L-proline; CHP) and the collagen inhibitor L-azetidine-2-carboxylic acid were tested for cytotoxicity against a large panel of cell lines. In addition to a tetrazolium-based assay (MTT), cell cycle analysis (propidium iodide) and apoptosis assays (annexin V) were performed. Amino acid uptake was studied using radioactive proline and differentiation was assessed by measuring induction of alkaline phosphatase (ALP). Results: Antiproliferative activity of CHP was found in cell lines derived from colon carcinomas, pancreatic, prostatic, and breast cancer as well as osteosarcoma and glioma (IC50: 35 - 175 μg/ml) and increased activity was detected under acidic extracellular conditions prevailing in solid tumors. Cell cycle arrest was found preferentially in G2M and apoptotic cells were detectable following prolonged exposure (7 - 10 days). CHP treatment of 2 colon carcinoma cell lines (HT29, CaCo-2) resulted in induction of the differentiation marker ALP. Proline competition tests indicated uptake of CHP via alpha-(methylamino)-isobutyric acid (MeAIB)-sensitive amino acid transport. Extensive testing of other cytotoxic proline-related compounds led to the characterization of N-methylated CHP derivatives, which show no interference with transport of proline, and proved to be capable of enhancing the CHP-induced toxicity by up to 30 %. Conclusions: Highly efficient proline analogs in combination exhibit considerable toxicity in vitro against diverse cell lines and should be studied in extended clinical CHP trials as agents modulating tumor cell growth by targeting ECM and/or critical proline residues of tumor-associated proteins. No significant financial relationships to disclose.

Identification of Residues Critical for Regulation of Protein Stability and the Transactivation Function of the Hypoxia-inducible Factor-1α by the von Hippel-Lindau Tumor Suppressor Gene Product

Under normoxic conditions the hypoxia-inducible factor-1alpha (HIF-1alpha) protein is targeted for degradation by the von Hippel-Lindau (pVHL) tumor suppressor protein acting as an E3 ubiquitin ligase. Binding of pVHL to HIF-1alpha is dependent on hydroxylation of specific proline residues by O(2)-dependent prolyl 4-hydroxylases. Upon exposure to hypoxia the hydroxylase activity is inhibited, resulting in stabilization of HIF-1alpha protein levels and activation of transcription of target genes. One of the two critical proline residues, Pro(563) in mouse HIF-1alpha, is located within a bifunctional domain, the N-terminal transactivation domain (N-TAD), which mediates both pVHL-dependent degradation at normoxia and transcriptional activation at hypoxia. Here we have identified two N-TAD residues, Tyr(564) and Ile(565), which, in addition to Pro(563), were critical for pVHL-mediated degradation at normoxia. We have also identified D568A/D569A/D570A, F571A, and L573A as mutations of the N-TAD that abrogated binding to pVHL both in vitro and in vivo, and constitutively stabilized N-TAD against degradation. Moreover, the mutations Y564G, L556A/L558A, and F571A/L573A drastically reduced the transactivation function of either the isolated N-TAD or full-length HIF-1alpha in hypoxic cells. Interestingly, the P563A mutant exhibited a constitutively active and potent transactivation function that was enhanced by functional interaction with the transcriptional coactivator protein CREB-binding protein. In conclusion, we have identified by mutation analysis several residues that are critical for either one or both of the interdigitated and conditionally regulated degradation and transactivation functions of the N-TAD of HIF-1alpha.

Autoinhibition of Mixed Lineage Kinase 3 through Its Src Homology 3 Domain

Mixed lineage kinase 3 (MLK3) is a serine/threonine protein kinase that functions as a mitogen-activated protein kinase kinase kinase to activate the c-Jun NH(2)-terminal kinase pathway. MLK3 has also been implicated as an I kappa B kinase kinase in the activation of NF-kappa B. Amino-terminal to its catalytic domain, MLK3 contains a Src homology 3 (SH3) domain. SH3 domains harbor three highly conserved aromatic amino acids that are important for ligand binding. In this study, we mutated one of these corresponding residues within MLK3 to deliberately disrupt the function of its SH3 domain. This SH3-defective mutant of MLK3 exhibited increased catalytic activity compared with wild type MLK3 suggesting that the SH3 domain negatively regulates MLK3 activity. We report herein that the SH3 domain of MLK3 interacts with full-length MLK3, and we have mapped the site of interaction to a region between the zipper and the Cdc42/Rac interactive binding motif. Interestingly, the SH3-binding region contains not a proline-rich sequence but, rather, a single proline residue. Mutation of this sole proline abrogates SH3 binding and increases MLK3 catalytic activity. Taken together, these data demonstrate that MLK3 is autoinhibited through its SH3 domain. The critical proline residue in the SH3-binding site of MLK3 is conserved in the closely related family members, MLK1 and MLK2, suggesting a common autoinhibitory mechanism among these kinases. Our study has revealed the first example of SH3 domain-mediated autoinhibition of a serine/threonine kinase and provides insight into the regulation of the mixed lineage family of protein kinases.

Molecular characterization of four induced alleles at the Ednrb locus.

The piebald locus on mouse chromosome 14 encodes the endothelin-B receptor (EDNRB), a G protein-coupled, seven-transmembrane domain protein, which is required for neural crest-derived melanocyte and enteric neuron development. A spontaneous null allele of Ednrb results in homozygous mice that are predominantly white and die as juveniles from megacolon. To identify the important domains for EDNRB function, four recessive juvenile lethal alleles created by either radiation or chemical mutagens (Ednrb27Pub, Ednrb17FrS, Ednrb1Chlc, and Ednrb3Chlo) were examined at the molecular level. Ednrb27Pub mice harbor a mutation at a critical proline residue in the fifth transmembrane domain of the EDNRB protein. A gross genomic alteration within the Ednrb gene in Ednrb3Chlo results in the production of aberrantly sized transcripts and no authentic Ednrb mRNA. Ednrb17FrS mice exhibited a decreased level of Ednrb mRNA, supporting previous observations that the degree of spotting in piebald mice is dependent on the amount of EDNRB expressed. Finally, no molecular defect was detected in Ednrb1Chlc mice, which produce normal levels of Ednrb mRNA in adult brain, suggesting that the mutation affects important regulatory elements that mediate the expression of the gene during development.

Direct Binding of the Proline-rich Region of Protein Tyrosine Phosphatase 1B to the Src Homology 3 Domain of p130Cas

Protein tyrosine phosphatase 1B (PTP1B) is an abundant intracellular enzyme that is thought to act as a negative regulator of certain signaling pathways. The C terminus of PTP1B contains two proline-rich regions which conform to the canonical class II Src homology 3 domain binding motif, Pro-X-X-Pro-X-Arg. In this study, we establish that PTP1B interacts with Crk, Grb2, and p130(Cas) in vitro and with at least one of these, p130(Cas), in intact cells. The interaction of PTP1B and p130(Cas) is independent of tyrosine phosphorylation but can be disrupted by replacing two critical proline residues in the proline-rich domain of PTP1B between amino acids 301 and 315. When wild-type PTP1B is expressed in 3Y1-v-crk cells, p130(Cas) shows substantial dephosphorylation, whereas the PTP1B proline mutant does not have this effect. In 3Y1 and 3Y1 v-crk-transformed fibroblasts, almost all of the total PTP1B and about 40% of total p130(Cas) co-sediment with membranes composed primarily of endoplasmic reticulum. These results suggest that the proline-rich domain between amino acids 301 and 315 in PTP1B binds Src homology 3-containing proteins and that p130(Cas) may be a physiological target of this phosphatase in cells.

Critical proline residues of the cytoplasmic domain of the IL-5 receptor alpha chain and its function in IL-5-mediated activation of JAK kinase and STAT5.

The high-affinity receptor (R) for IL-5 consists of a unique alpha chain (IL-5R alpha) and a beta chain (beta c) that is shared with the receptors for IL-3 and granulocyte macrophage colony stimulating factor (GM-CSF). We defined two regions of IL-5R alpha for the IL-5-induced proliferative response, the expression of nuclear proto-oncogenes, and the tyrosine phosphorylation of cellular proteins including beta c, SH2/SH3-containing proteins and JAK2 kinase. In the studies described here, we demonstrate that IL-5, IL-3 or GM-CSF stimulation induces the tyrosine phosphorylation of JAK2, and to a lesser extent JAK1, and of STAT5. Mutational analysis revealed that one of the proline residues, particularly Pro352 and Pro355, in the membrane-proximal proline-rich sequence (Pro352-Pro353-X-Pro355) of the cytoplasmic domain of IL-5R alpha is required for cell proliferation, and for both JAK1 and JAK2 activation. In addition, transfectants expressing chimeric receptors which consist of the extracellular domain of IL-5R alpha and the cytoplasmic domain of beta c responded to IL-5 for proliferation and tyrosine phosphorylation of JAK1. Intriguingly, electrophoretic mobility shift assay analysis revealed that STAT5 was activated in cells showing either JAK1 or JAK2 tyrosine phosphorylation. These results indicate that activation of JAK1, JAK2 and STAT5 is critical to coupling IL-5-induced tyrosine phosphorylation and ultimately mitogenesis, and that Pro352 and Pro355 in the proline-rich sequence appear to play more essential roles in cell growth and in both JAK1/STAT5 and JAK2/STAT5 activation than Pro353 does.