Tyrosine kinases in neutrophils

Outcome after radiofrequency thermocoagulation in patients with trigeminal neuralgia was assessed in a prospective, longitudinal study. Forty-eight consecutive patients with chronic facial pain presenting for surgery to a neurosurgeon were studied. Patients were assessed preoperatively by an independent clinician both clinically, and with the use of two questionnaires: the McGill Pain Questionnaire (MPQ) and the Hospital Anxiety and Depression (HAD) scale. From these assessments, two groups of patients were identified: 31 with pure trigeminal neuralgia (TN group) and 17 with trigeminal neuralgia together with atypical facial pain and mixed trigeminal neuralgia (MTN group). All underwent radiofrequency thermocoagulation at the level of the Gasserian ganglion. Patients were reviewed by the same clinician 3 months later and then followed up by a self-administered questionnaire at 6 months, 1 year, 2 years and 3 years. The mean follow-up time was 30+/-12 months. The mean time to recurrence of pain was 40 months for the TN group and 36 months for the MTN group. Depression and anxiety dropped more significantly post-operatively in the TN group than the MTN group. TN group were more satisfied with their outcome, complained of fewer complications and were more willing to have repeat surgery if necessary than patients in MTN group. The number and severity of complications varied at different time points. Careful selection of patients for surgery using objective assessments will decrease morbidity and improve satisfaction. Physical morbidity and recurrence rates are insufficient to gauge outcomes. Psychological, sociological and patients' views must be included in evaluations.     

Tyrosine phosphorylation and src family kinases control keratinocyte cell-cell adhesion

In their progression from the basal to upper differentiated layers of the epidermis, keratinocytes undergo significant structural changes, including establishment of close intercellular contacts. An important but so far unexplored question is how these early structural events are related to the biochemical pathways that trigger differentiation. We show here that beta-catenin, gamma-catenin/plakoglobin, and p120-Cas are all significantly tyrosine phosphorylated in primary mouse keratinocytes induced to differentiate by calcium, with a time course similar to that of cell junction formation. Together with these changes, there is an increased association of alpha-catenin and p120-Cas with E-cadherin, which is prevented by tyrosine kinase inhibition. Treatment of E-cadherin complexes with tyrosine-specific phosphatase reveals that the strength of alpha-catenin association is directly dependent on tyrosine phosphorylation. In parallel with the biochemical effects, tyrosine kinase inhibition suppresses formation of cell adhesive structures, and causes a significant reduction in adhesive strength of differentiating keratinocytes. The Fyn tyrosine kinase colocalizes with E-cadherin at the cell membrane in calcium-treated keratinocytes. Consistent with an involvement of this kinase, fyn-deficient keratinocytes have strongly decreased tyrosine phosphorylation levels of beta- and gamma-catenins and p120-Cas, and structural and functional abnormalities in cell adhesion similar to those caused by tyrosine kinase inhibitors. Whereas skin of fyn-/- mice appears normal, skin of mice with a disruption in both the fyn and src genes shows intrinsically reduced tyrosine phosphorylation of beta-catenin, strongly decreased p120-Cas levels, and important structural changes consistent with impaired keratinocyte cell adhesion. Thus, unlike what has been proposed for oncogene-transformed or mitogenically stimulated cells, in differentiating keratinocytes tyrosine phosphorylation plays a positive role in control of cell adhesion, and this regulatory function appears to be important both in vitro and in vivo.     

A family of phase-variable restriction enzymes with differing specificities generated by high-frequency gene rearrangements

The hsd genes of Mycoplasma pulmonis encode restriction and modification enzymes exhibiting a high degree of sequence similarity to the type I enzymes of enteric bacteria. The S subunits of type I systems dictate the DNA sequence specificity of the holoenzyme and are required for both the restriction and the modification reactions. The M. pulmonis chromosome has two hsd loci, both of which contain two hsdS genes each and are complex, site-specific DNA inversion systems. Embedded within the coding region of each hsdS gene are a minimum of three sites at which DNA inversions occur to generate extensive amino acid sequence variations in the predicted S subunits. We show that the polymorphic hsdS genes produced by gene rearrangement encode a family of functional S subunits with differing DNA sequence specificities. In addition to creating polymorphisms in hsdS sequences, DNA inversions regulate the phase-variable production of restriction activity because the other genes required for restriction activity (hsdR and hsdM) are expressed only from loci that are oriented appropriately in the chromosome relative to the hsd promoter. These data cast doubt on the prevailing paradigms that restriction systems are either selfish or function to confer protection from invasion by foreign DNA.     

Engineering protein kinases with distinct nucleotide specificities and inhibitor sensitivities by mutation of a single amino acid

A major goal of signal transduction research is to identify the substrates and roles of the many protein kinases. The task might be simplified by the discovery that the mutation of a single amino acid dramatically alters the nucleotide specificity of protein kinases and their inhibition by a particular class of anti-inflammatory drug.     

SMART, a simple modular architecture research tool: identification of signaling domains

Accurate multiple alignments of 86 domains that occur in signaling proteins have been constructed and used to provide a Web-based tool (SMART: simple modular architecture research tool) that allows rapid identification and annotation of signaling domain sequences. The majority of signaling proteins are multidomain in character with a considerable variety of domain combinations known. Comparison with established databases showed that 25% of our domain set could not be deduced from SwissProt and 41% could not be annotated by Pfam. SMART is able to determine the modular architectures of single sequences or genomes; application to the entire yeast genome revealed that at least 6.7% of its genes contain one or more signaling domains, approximately 350 greater than previously annotated. The process of constructing SMART predicted (i) novel domain homologues in unexpected locations such as band 4.1-homologous domains in focal adhesion kinases; (ii) previously unknown domain families, including a citron-homology domain; (iii) putative functions of domain families after identification of additional family members, for example, a ubiquitin-binding role for ubiquitin-associated domains (UBA); (iv) cellular roles for proteins, such predicted DEATH domains in netrin receptors further implicating these molecules in axonal guidance; (v) signaling domains in known disease genes such as SPRY domains in both marenostrin/pyrin and Midline 1; (vi) domains in unexpected phylogenetic contexts such as diacylglycerol kinase homologues in yeast and bacteria; and (vii) likely protein misclassifications exemplified by a predicted pleckstrin homology domain in a Candida albicans protein, previously described as an integrin.     

Analysis of signaling protein kinases in human colon or colorectal carcinomas

Extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase (JNK) mitogen-activated protein (MAP) kinases are highly activated in an in vivo rat model of colorectal carcinogenesis. In addition, other protein kinases such as c-Src and c-Yes have been shown to be up-regulated in some human colon cancers. To evaluate the activity of these kinases in human colorectal carcinomas, we examined colon cancers and adjacent normal intestinal mucosa from 11 patients. Moderate increases in ERK and JNK activities, in addition to up-regulation of c-Src, p125FAK, and tyrosine-phosphorylated proteins, were observed in a subset of the colorectal carcinomas. There was a significant correlation found between levels of c-Src, p125FAK, and tyrosine-phosphorylated proteins, as well as between c-Src protein levels and JNK activity. This is the first report that examines several different kinases as markers to characterize colorectal cancers in the same carcinoma sample, allowing the determination of correlations between markers in the same tumors.     

Biochemical properties of two protein kinases involved in disease resistance signaling in tomato

In tomato plants, resistance to bacterial speck disease is mediated by a phosphorylation cascade, which is triggered by the specific recognition between the plant serine/threonine protein kinase Pto and the bacterial AvrPto protein. In the present study, we investigated in vitro biochemical properties of Pto, which appears to function as an intracellular receptor for the AvrPto signal molecule. Pto and its downstream effector Pti1, which is also a serine/threonine protein kinase, were expressed in Escherichia coli as maltose-binding protein and glutathione S-transferase fusion proteins, respectively. The two kinases each autophosphorylated at multiple sites as determined by phosphopeptide mapping. In addition, Pto and Pti1 autophosphorylation occurred via an intramolecular mechanism, as their specific activity was not affected by their molar concentration in the assay. Moreover, an active glutathione S-transferase-Pto fusion failed to phosphorylate an inactive maltose-binding protein-Pto(K69Q) fusion excluding an intermolecular mechanism of phosphorylation for Pto. Pti1 phosphorylation by Pto was also characterized and found to occur with a Km of 4.1 microM at sites similar to those autophosphorylated by Pti1. Pto and the product of the recessive allele pto phosphorylated Pti1 at similar sites, as observed by phosphopeptide mapping. This suggests that the inability of the kinase pto to confer resistance to bacterial speck disease in tomato is not caused by altered recognition specificity for Pti1 phosphorylation sites.     

Interaction of the maize and Arabidopsis kinase interaction domains with a subset of receptor-like protein kinases: implications for transmembrane signaling in plants

The American population is aging rapidly and individuals are living longer. Yet Americans are saving less and older workers are leaving the labor force at younger and younger ages. The accelerated drop in labor force participation corresponds roughly to the introduction of Social Security and the adaptation of employer-provided pension plans. I have illustrated that Social Security and employer-provided pension plans provide substantial incentive to leave the labor force early. The quantitative effect of this inducement is illustrated by simulating the effects of changes in pension plan and Social Security provisions on the retirement decisions of employees in a large firm, who are covered by a typical defined benefit pension plan. Scheduled Social Security changes would have little effect on the retirement decisions of employees with a typical defined benefit pension plan like the one considered here. But if the pension plan provisions were changed to correspond to the Social Security changes, the effect would be very large. And, although not contemplated by current legislation; it is clear that an increase in the Social Security early retirement age would have a substantial effect on the early retirement rates of the large number of employees not covered by a pension plan.     

Selective neural cell adhesion molecule signaling by Src family tyrosine kinases and tyrosine phosphatases

Nerve growth cone guidance is a highly complex feat, involving coordination of cell adhesion molecules, trophic factor gradients, and extracellular matrix proteins. While navigating through the developing nervous system, the growth cone must integrate diverse environmental signals into a singular response. The repertoire of growth cone responses to these extracellular cues includes axonal growth, fasciculation, and synaptic stabilization, which are achieved through dynamic changes in the cytoskeleton and modulation of gene expression. It has become evident that interactions between cell adhesion molecules can activate intracellular signaling pathways in neurons. Such signaling pathways are just beginning to be defined for the axonal growth promoting molecules L1 and NCAM which are members of the immunoglobulin (Ig) superfamily. Recent findings have revealed that L1 and NCAM induce neurite outgrowth by activating intracellular signaling pathways in the growth cone mediated by two different members of the src family of nonreceptor protein tyrosine kinases (PTKs), pp60(c-src) and p59(fyn5,6). Growth cones display diverse morphologies and variable motility on these different cell adhesion molecules, which are likely to be generated by src kinases. In this review we will address novel features of nonreceptor PTKs of the src family which dictate their distinctive molecular interactions with cell adhesion molecules and signaling components.     

Differential expression of ZAP-70 and Syk protein tyrosine kinases, and the role of this family of protein tyrosine kinases in TCR signaling

TCR stimulation results in the tyrosine phosphorylation of a number of cellular substrates. We have recently identified a 70-kDa protein tyrosine kinase, ZAP-70, which associates with the human TCR zeta-chain after TCR stimulation. We report here the isolation and sequence of a cDNA clone that encodes murine ZAP-70. Murine and human ZAP-70 share 93% amino acid identity and are homologous to the 72-kDa protein tyrosine kinase Syk. Syk has been implicated in the signal transduction pathways of the B cell membrane Ig and high affinity IgE receptors, Fc epsilon RI. In addition, we examined the tissue distribution of ZAP-70 and Syk in human and murine thymocyte subsets, B cells, and peripheral T cell subsets. ZAP-70 protein is expressed in all major thymocyte populations, with the level of expression being comparable to that found in both CD4+ and CD8+ peripheral T cells. Although Syk protein is also present in all thymocyte subsets, expression of Syk protein is down-regulated threefold to fourfold in peripheral T cells. In contrast to ZAP-70, expression of Syk is 12- to 15-fold higher in peripheral B cells when compared with peripheral T cells. In addition, whereas T cell stimulation results in down-regulation of Lck, no significant change in ZAP-70 or Syk protein is detected. Finally, we provide evidence that both ZAP-70 and Syk can associate with the TCR after TCR stimulation. With the use of a heterologous expression system, we show that, like ZAP-70, Syk is dependent upon a Src-family protein tyrosine kinase for association with the phosphorylated zeta-chain. Thus, the differential expression of these kinases suggests the possibility of different roles for ZAP-70 and Syk in TCR signaling and thymic development.     

A structural basis for substrate specificities of protein Ser/Thr kinases: primary sequence preference of casein kinases I and II, NIMA, phosphorylase kinase, calmodulin-dependent kinase II, CDK5, and Erk1

We have developed a method to study the primary sequence specificities of protein kinases by using an oriented degenerate peptide library. We report here the substrate specificities of eight protein Ser/Thr kinases. All of the kinases studied selected distinct optimal substrates. The identified substrate specificities of these kinases, together with known crystal structures of protein kinase A, CDK2, Erk2, twitchin, and casein kinase I, provide a structural basis for the substrate recognition of protein Ser/Thr kinases. In particular, the specific selection of amino acids at the +1 and -3 positions to the substrate serine/threonine can be rationalized on the basis of sequences of protein kinases. The identification of optimal peptide substrates of CDK5, casein kinases I and II, NIMA, calmodulin-dependent kinases, Erk1, and phosphorylase kinase makes it possible to predict the potential in vivo targets of these kinases.     

Growth factor receptor tyrosine kinases acutely regulate neuronal sodium channels through the src signaling pathway

Growth factor receptor tyrosine kinase (RTK)-activated signaling pathways are well established regulators of neuronal growth and development, but whether these signals provide mechanisms for acute modulation of neuronal activity is just beginning to be addressed. We show in pheochromocytoma (PC12) cells that acute application of ligands for both endogenous RTKs [trkA, basic FGF (bFGF) receptor, and epidermal growth factor (EGF) receptor] and ectopically expressed platelet-derived growth factor (PDGF) receptors rapidly inhibits whole-cell sodium channel currents, coincident with a hyperpolarizing shift in the voltage dependence of inactivation. Sodium channel inhibition by trkA and PDGF receptors is mutually occlusive, suggestive of a common signal transduction mechanism. Furthermore, specific inhibitors for trkA and PDGF RTK activities abrogate sodium channel inhibition in response to NGF and PDGF, respectively, showing that the intrinsic RTK activity of these receptors is necessary for sodium channel inhibition. Use of PDGF receptor mutants deficient for specific signaling activities demonstrated that this inhibition is dependent on RTK interaction with Src but not with other RTK-associated signaling molecules. Inhibition was also compromised in cells expressing dominant-negative Ras. These results suggest a possible mechanism for acute physiological actions of RTKs, and they indicate regulatory functions for Ras and Src that may complement the roles of these signaling proteins in long-term neuronal regulation.