Dysregulated Src upregulation of NMDA receptor activity: a common link in chronic pain and schizophrenia

Upregulation of N-methyl-D-aspartate (NMDA) receptor function by the nonreceptor protein tyrosine kinase Src has been implicated in physiological plasticity at glutamatergic synapses. Here, we highlight recent findings suggesting that aberrant Src upregulation of NMDA receptors may also be key in pathophysiological conditions. Within the nociceptive processing network in the dorsal horn of the spinal cord, pathologically increased Src upregulation of NMDA receptors is critical for pain hypersensitivity in models of chronic inflammatory and neuropathic pain. On the other hand, in the hippocampus and prefrontal cortex, the physiological upregulation of NMDA receptors by Src is blocked by neuregulin 1-ErbB4 signaling, a pathway that is genetically implicated in the positive symptoms of schizophrenia. Thus, either over-upregulation or under-upregulation of NMDA receptors by Src may lead to pathological conditions in the central nervous system. Therefore, normalizing Src upregulation of NMDA receptors may be a novel therapeutic approach for central nervous system disorders, without the deleterious consequences of directly blocking NMDA receptors.     

Phosphoryl transfer step in the C-terminal Src kinase controls Src recognition

All members of the Src family of nonreceptor protein tyrosine kinases are phosphorylated and subsequently down-regulated by the C-terminal Src kinase, Csk. Although the recognition of Src protein substrates is essential for a diverse set of signaling events linked to cellular growth and differentiation, the factors controlling this critical protein-protein interaction are not well known. To understand how Csk recognizes Src, the chemical/physical events that modulate apparent substrate affinity and turnover were investigated. Src is phosphorylated in a biphasic manner in rapid quench flow experiments, suggesting that the phosphoryl transfer step is fast and highly favorable and does not limit overall turnover. As opposed to other kinase-substrate pairs, turnover is not limited by the physical release of ADP based on stopped-flow fluorescence and catalytic trapping experiments, suggesting that other steps control net phosphorylation. The K(d) for Src is considerably larger than the K(m) based on single turnover kinetic and equilibrium sedimentation experiments. Taken together, the data are consistent with a mechanism whereby Csk achieves a low K(m) for the substrate Src, not by stabilizing protein-protein interactions but rather by facilitating a fast phosphoryl transfer step. In this manner, the phosphoryl transfer step functions as a chemical clamp facilitating substrate recognition.     

Src kinase activation: A switched electrostatic network

Src tyrosine kinases are essential in numerous cell signaling pathways, and improper functioning of these enzymes has been implicated in many diseases. The activity of Src kinases is regulated by conformational activation, which involves several structural changes within the catalytic domain (CD): the orientation of two lobes of CD; rearrangement of the activation loop (A-loop); and movement of an alpha-helix (alphaC), which is located at the interface between the two lobes, into or away from the catalytic cleft. Conformational activation was investigated using biased molecular dynamics to explore the transition pathway between the active and the down-regulated conformation of CD for the Src-kinase family member Lyn kinase, and to gain insight into the interdependence of these changes. Lobe opening is observed to be a facile motion, whereas movement of the A-loop motion is more complex requiring secondary structure changes as well as communication with alphaC. A key result is that the conformational transition involves a switch in an electrostatic network of six polar residues between the active and the down-regulated conformations. The exchange between interactions links the three main motions of the CD. Kinetic experiments that would demonstrate the contribution of the switched electrostatic network to the enzyme mechanism are proposed. Possible implications for regulation conferred by interdomain interactions are also discussed.     

Src signaling in cancer invasion

Src is a non‐receptor cytoplasmic tyrosine kinase which becomes activated following the stimulation of plasma membrane receptors including receptor tyrosine kinases and integrins, and is an indispensable player of multiple physiological homeostatic pathways. Once activated, Src is the starting point for several biochemical cascades that thereby propagate signals generated extracellularly along intracellular interconnected transduction pathways. Src transmits signals promoting cell survival and mitogenesis and, in addition, exerts a profound effect on the reorganization of the cytoskeleton and the adhesion systems that underpin cell migration and invasion. Because increased activity of Src is a frequent occurrence in many types of human cancer, and because there is evidence of a prominent role of Src in invasion and in other tumor progression‐related events such as epithelial–mesenchymal transition (EMT) and development of metastasis, inhibitors targeting Src are being viewed as promising drugs for cancer therapy. J. Cell. Physiol. 223: 14–26, 2010. © 2009 Wiley‐Liss, Inc.     

Src kinase signaling in leukaemia

Role of Src kinases in acute lymphoblastic leukaemia has been recently demonstrated in leukaemia mouse model. Retained activation of Src kinases by the BCR-ABL oncoprotein in leukaemic cells following inhibition of BCR-ABL kinase activity by imatinib indicates that Src activation by BCR-ABL is independent of BCR-ABL kinase activity and provides an explanation for reduced effectiveness of the BCR-ABL kinase activity inhibitors in Philadelphia chromosome-positive acute lymphoblastic leukaemia. Simultaneous inhibition of kinase activity of both BCR-ABL and Src kinases results in long-term survival of mice with acute lymphoblastic leukaemia. Leukaemic stem cells exist in acute lymphoblastic leukaemia, and complete eradication of this group of cells would provide a curative therapy for this disease.     

Src deficiency or blockade of Src activity in mice provides cerebral protection following stroke

Vascular endothelial growth factor (VEGF), an angiogenic factor produced in response to ischemic injury, promotes vascular permeability (VP). Evidence is provided that Src kinase regulates VEGF-mediated VP in the brain following stroke and that suppression of Src activity decreases VP thereby minimizing brain injury. Mice lacking pp60c-src are resistant to VEGF-induced VP and show decreased infarct volumes after stroke whereas mice deficient in pp59c-fyn, another Src family member, have normal VEGF-mediated VP and infarct size. Systemic application of a Src-inhibitor given up to six hours following stroke suppressed VP protecting wild-type mice from ischemia-induced brain damage without influencing VEGF expression. This was associated with reduced edema, improved cerebral perfusion and decreased infarct volume 24 hours after injury as measured by magnetic resonance imaging and histological analysis. Thus, Src represents a key intermediate and novel therapeutic target in the pathophysiology of cerebral ischemia where it appears to regulate neuronal damage by influencing VEGF-mediated VP.     

A crystal milestone: The structure of regulated Src

The viral and cellular forms of the Src protein tyrosine kinases take a prototypic role in oncology and signal transduction research, by virtue of being holders of an impressive number of ‘firsts’. Our understanding of the biochemistry and physiology of Src has therefore always been used as a reference for our general advancement in the field of protein phosphorylation and growth control. The recent solution of the crystal structure of two members of the Src family^(1,2) represents a milestone in these disciplines and, as usual, provides a general lookout post for developments to come.     

Reversion-induced LIM interaction with Src reveals a novel Src inactivation cycle

Aberrant Src activation plays prominent roles in cancer progression. However, how Src is activated in cancer cells is largely unknown. Genetic Src-activating mutations are rare and, therefore, are insufficient to account for Src activation commonly found in human cancers. In this study, we show that reversion-induced LIM (RIL), which is frequently lost in colon and other cancers as a result of epigenetic silencing, suppresses Src activation. Mechanistically, RIL suppresses Src activation through interacting with Src and PTPL1, allowing PTPL1-dependent dephosphorylation of Src at the activation loop. Importantly, the binding of RIL to Src is drastically reduced upon Src inactivation. Our results reveal a novel Src inactivation cycle in which RIL preferentially recognizes active Src and facilitates PTPL1-mediated inactivation of Src. Inactivation of Src, in turn, promotes dissociation of RIL from Src, allowing the initiation of a new Src inactivation cycle. Epigenetic silencing of RIL breaks this Src inactivation cycle and thereby contributes to aberrant Src activation in human cancers.     

Tubulogenesis: Src42A goes to great lengths in tube elongation

New work shows the instructive role of Src42A kinase in tube size regulation. By inducing polarized cell-shape changes, Src42A promotes tube elongation in the Drosophila tracheal system.     

Identification of a link between the SAMP repeats of adenomatous polyposis coli tumor suppressor and the Src homology 3 domain of DDEF

The adenomatous polyposis coli (APC) tumor suppressor protein is a multifunctional protein with a well characterized role in the Wnt signal transduction pathway and in cytoskeletal regulation. The SAMP repeats region of APC, an Axin-binding site, is known to be important for tumor suppression and for the developmental function of APC. We performed a yeast two-hybrid screening using the first SAMP motif-containing region of Xenopus APC as bait and obtained several SAMP binding candidates including DDEF2 (development and differentiation enhancing factor 2), which is an ADP-ribosylation factor (Arf) GTPase-activating protein (GAP (ArfGAP)) involved in the regulation of focal adhesions. In vitro and in cells the Src homology 3 (SH3) domain of DDEF2 and its close homolog, DDEF1, are associated with the SAMP motif of APC competitively with Axin1. Moreover, NMR chemical shift perturbation experiments revealed that the SAMP motif interacts at the same surface of the SH3 domain of DDEF as the known SH3 binding motif, PXXP. When fluorescent protein-tagged APC and DDEF are expressed in Xenopus A6 cells, co-localization at microtubule ends is observed. Overexpression and RNA interference experiments indicate that APC and DDEFs cooperatively regulate the distributions of microtubules and focal adhesions. Our findings reveal that the SAMP motif of APC specifically binds to the SH3 domains of DDEFs, providing new insights into the functions of APC in cell migration.     

A phosphotyrosine displacement mechanism for activation of Src by PTPalpha

Protein tyrosine phosphatase alpha (PTPalpha) is believed to dephosphorylate physiologically the Src proto-oncogene at phosphotyrosine (pTyr)527, a critical negative-regulatory residue. It thereby activates Src, and PTPalpha overexpression neoplastically transforms NIH 3T3 cells. pTyr789 in PTPalpha is constitutively phosphorylated and binds Grb2, an interaction that may inhibit PTPalpha activity. We show here that this phosphorylation also specifically enables PTPalpha to dephosphorylate pTyr527. Tyr789-->Phe mutation abrogates PTPalpha-Src binding, dephosphorylation of pTyr527 (although not of other substrates), and neoplastic transformation by overexpressed PTPalpha in vivo. We suggest that pTyr789 enables pTyr527 dephosphorylation by a pilot binding with the Src SH2 domain that displaces the intramolecular pTyr527-SH2 binding. Consistent with model predictions, we find that excess SH2 domains can disrupt PTPalpha-Src binding and can block PTPalpha-mediated dephosphorylation and activation in proportion to their affinity for pTyr789. Moreover, we show that, as predicted by the model, catalytically defective PTPalpha has reduced Src binding in vivo. The displacement mechanism provides another potential control point for physiological regulation of Src-family signal transduction pathways.     

Phosphorylation of a Src kinase at the autophosphorylation site in the absence of Src kinase activity

Exposure of cells to oxidants increases the phosphorylation of the Src family tyrosine protein kinase Lck at Tyr-394, a conserved residue in the activation loop of the catalytic domain. Kinase-deficient Lck expressed in fibroblasts that do not express any endogenous Lck has been shown to be phosphorylated at Tyr-394 following H(2)O(2) treatment to an extent indistinguishable from that seen with wild type Lck. This finding indicates that a kinase other than Lck itself is capable of phosphorylating Tyr-394. Because fibroblasts express other Src family members, it remained to be determined whether the phosphorylation of Tyr-394 was carried out by another Src family kinase or by an unrelated tyrosine protein kinase. We examined here whether Tyr-394 in kinase-deficient Lck was phosphorylated following exposure of cells devoid of endogenous Src family kinase activity to H(2)O(2). Strikingly, treatment of such cells with H(2)O(2) led to the phosphorylation of Tyr-394 to an extent identical to that seen with wild type Lck, demonstrating that Src family kinases are not required for H(2)O(2)-induced phosphorylation of Lck. Furthermore, this efficient phosphorylation of Lck at Tyr-394 in non-lymphoid cells suggests the existence of an ubiquitous activator of Src family kinases.     

Biochemical studies in schizophrenia: A review

CATHOLIC VIEWPOINT ON OVERPOPULATION: Anthony Zimmerman, S.V.D. Hanover House, New York, 1961, 214 pp., $3.50.

MANKIND EVOLVING: Theodosius Dobzhansky. Yale University Press, New Haven, 1962, xiii + 381 pp., $7.50.

THE SEARCH FOR A COMMON LEARNING: General Education, 1800–1960: Russell Thomas. McGraw‐Hill, New York, 1962, 324 pp., $6.95.

THE WORLD ROLE OF THE UNIVERSITIES: Edward W. Weidner. McGraw‐Hill, New York, 1962, 366 pp., $3.65.

RESEARCH IN FAMILY PLANNING: C. V. Kiser, ed. Princeton University Press, 1962, Princeton, pp. 662, $12.50.     

Bacteria—A link among ecosystem constituents

Microbial ecology has undergone a revolution over the past two decades due to the numerous innovations in techniques, allowing bacteria to be detected more accurately by direct means. Thus, bacterial life can be distinguishedin situ by direct counting under epifluorescence microscopy; automatically counting and sizing by image analyzer equipped with epifluorescence microscopy or by use of flow cytometry; specific radioisotope techniques; and molecular techniques to detect specific taxa. All of these approaches do not require cultivation, which provides a biased view of bacterial communities in nature. Bacteria are abundant in aquatic environments and play important roles as links between dissolved nutrients and the grazers in the food web. The new techniques allow an evaluation of bacterial population dynamics and function in relation to other organisms of higher trophic levels. This mini review aims to show briefly the state-of-the-art in microbial ecology for ecologists concerned with organisms other than microbes, in order to develop further intensive study together with microbial ecologists.     

A physics-based link model for tree vibrations

• Premise of the study: A new mathematical model for the vibration of trees is presented for developing a more thorough understanding of the underlying structure of the response. It may be used, for example, to assess the stability of a tree or to interpret experimental data. • Methods: A model is developed for the motion of the trunk and its N number of branches. The spatial distribution and initial orientation of the branches are left for the user to prescribe. A Newtonian analysis yields (N + 1) nonlinear, coupled differential equations that, when solved, describe the response of the trunk and each branch. After the model is linearized near equilibrium, the natural frequencies and vibration mode shapes are found. Closed-form expressions for the response (i.e., the actual time histories) are then obtained using modal analysis. Numerical solutions are also found; these are used to validate the analytical solutions and to serve as a means for considering large amplitude vibrations. • Key results: A new physics-based model is described. For small motion, the tree response may be constructed from the individual mode shapes and frequencies. Also demonstrated are the limitations of the linear theory as well as numerical solutions that can be obtained when trunk/branch amplitudes are large. • Conclusions: The model presented here incorporates critical physics into a model that describes tree vibrations. It also sheds light on the underlying structure of the vibration response in terms of the modal nature of the solution. Limitations to the linear solutions are demonstrated and discussed.