Neurones express high levels of a structurally modified, activated form of pp60c-src

Brugge, Joan S.; Cotton, P C; Queral, A. E.; Barrett, John N.; Nonner, Doris; Keane, Robert W.

doi:10.1038/316554a0

Cited by 364 publications

(245 citation statements)

References 31 publications

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“…In addition to c-Src, neurons also express high levels of n-Src, a neuron-specific splice variant that differs by only a 6-aa insert within the SH3 domain (9,23). In contrast to the results obtained with the GST-c-Src-SH3 domain, overlay assay failed to detect any specific interactions of the GST-n-Src-SH3 domain with nerve terminal proteins (not shown).…”

Section: Resultscontrasting

confidence: 46%

“…In addition to c-Src, the expression of two neuron-specific Src isoforms has been detected in mammalian brain (9,22). These isoforms arise from alternative splicing events that direct the insertion of either one or both of two short exons, termed the NI exon (18 nt, encoding a 6-aa insert) and the NII exon (33 nt, encoding an 11-aa insert), into the region encoding the SH3 domain (22,23).…”

Section: Discussionmentioning

confidence: 99%

“…Antibodies against synapsin I, dynamin, synaptojanin, and glutathione S-transferase (GST) have been described (1,7). The SH3 domain fusion protein constructs were generously provided by the following individuals: pGEX-c-Src-SH3 domain (8), I. Gout (Ludwig Institute, London); pGEX-n-Src-SH3 domain (9), J. S. Brugge (Ariad Pharmaceuticals, Cambridge, MA); pGEX-Grb2 (10), J. Schlessinger (New York University Medical Center, New York). Purified recombinant human c-Src (900,000 units͞mg) was purchased from Upstate Biotechnology (Lake Placid, NY).…”

Section: Methodsmentioning

confidence: 99%

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Synapsin I interacts with c-Src and stimulates its tyrosine kinase activity

Onofri

Giovedì

Vaccaro

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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Synapsin I is a synaptic vesicle-associated phosphoprotein that has been implicated in the formation of presynaptic specializations and in the regulation of neurotransmitter release. The nonreceptor tyrosine kinase c-Src is enriched on synaptic vesicles, where it accounts for most of the vesicle-associated tyrosine kinase activity. Using overlay, affinity chromatography, and coprecipitation assays, we have now shown that synapsin I is the major binding protein for the Src homology 3 (SH3) domain of c-Src in highly purified synaptic vesicle preparations. The interaction was mediated by the proline-rich domain D of synapsin I and was not significantly affected by stoichiometric phosphorylation of synapsin I at any of the known regulatory sites. The interaction of purified c-Src and synapsin I resulted in a severalfold stimulation of tyrosine kinase activity and was antagonized by the purified c-Src-SH3 domain. Depletion of synapsin I from purified synaptic vesicles resulted in a decrease of endogenous tyrosine kinase activity. Portions of the total cellular pools of synapsin I and Src were coprecipitated from detergent extracts of rat brain synaptosomal fractions using antibodies to either protein species. The interaction between synapsin I and c-Src, as well as the synapsin I-induced stimulation of tyrosine kinase activity, may be physiologically important in signal transduction and in the modulation of the function of axon terminals, both during synaptogenesis and at mature synapses.Synapsin I is the major synaptic vesicle protein that binds to the Src homology 3 (SH3) domains of the adapter protein Grb2 in vitro (1). This interaction is mediated by domain D of synapsin I, a 23-kDa proline-rich, strongly basic domain located in the COOH-terminal portion of synapsin I (2). It seemed possible that domain D or other proline-rich regions in synapsin I might interact with other SH3 domain-containing proteins within the nerve terminal and that these interactions might have a physiological role in presynaptic function. One such candidate, the SH3 domain-containing nonreceptor tyrosine kinase c-Src, is expressed at high levels in postmitotic neurons and is enriched on synaptic vesicles, where it accounts for most of the vesicle-associated tyrosine kinase activity (3-6). Using purified components in vitro, we now report that a domain Dmediated interaction of synapsin I with the SH3 domain of c-Src results in a 5-fold activation of the catalytic activity of c-Src. We also present studies employing a variety of biochemical techniques that serve to further characterize the specificity, subcellular location, regulation, and potential functional consequences of the c-Src͞synapsin I interaction.

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Section: Resultscontrasting

confidence: 46%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Synapsin I interacts with c-Src and stimulates its tyrosine kinase activity

Onofri

Giovedì

Vaccaro

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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“…However, the tissue distribution of pp6Oc-src expression suggests that this is not its function; those cells which express the highest levels of pp60csrc do not divide. For example, postmitotic differentiated or differentiating neurons express high levels of both c-src proteins (1,3,7,8,17,21). Mammalian platelets, terminally differentiated cells with no growth potential, express still higher levels of the smaller c-src protein (9).…”

mentioning

confidence: 99%

“…Little is known about the physiological function of either c-src protein. A number of other proto-oncogene products are involved in the transduction of normal mitogenic signals (e.g., c-erbB [epidermal growth factor receptor], c-fms [colony-stimulating factor 1 receptor], c-sis [platelet-derived growth factor]). However, the tissue distribution of pp6Oc-src expression suggests that this is not its function; those cells which express the highest levels of pp60csrc do not divide.…”

mentioning

confidence: 99%

Platelet tyrosine-specific protein phosphorylation is regulated by thrombin.

Ferrell¹,

Martin²

1988

Mol. Cell. Biol.

230

120

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Intact human platelets, terminally differentiated cells with no growth potential, were found to possess unusually high levels of tyrosine-specific protein phosphorylation. The physiological platelet activator thrombin transiently elevated platelet phosphotyrosine content, apparently through stimulation of one or more tyrosine-specific protein kinases. Immunoblotting with antiphosphotyrosine antiserum showed that thrombin caused dramatic changes in the tyrosine phosphorylation of a number of individual protein bands and that these changes occurred in three distinct temporal waves. Most but not all of the protein bands phosphorylated at tyrosine in response to thrombin were also tyrosine phosphorylated in response to chilling or the combination of ionophore A23187 and tetradecanoylphorbol acetate. Thrombin stimulated the phosphorylation of the tyrosine kinase pp6OC-S', primarily at Ser-12 and Tyr-527, although the effects of these phosphorylations on platelet pp60csrC function were not apparent. Together, these results suggest that tyrosine-specific protein kinases of uncertain identity are involved in signal transduction in platelets.The phosphorylation of proteins at tyrosine residues is implicated in normal and abnormal cell growth: several growth factor receptors are tyrosine-specific protein kinases, as are the transforming proteins of one class of acutely oncogenic retroviruses (13). The best-studied such retrovirus is Rous sarcoma virus, whose transforming gene v-src was derived from a cellular tyrosine kinase gene, c-src (22, 23). The protein-tyrosine kinase activity of the v-src gene product pp60vsrc has been activated by a C-terminal substitution and scattered point mutations (12). The transforming capacity of pp60v-src results, at least in part, from its increased kinase activity.Through alternative splicing, the c-src gene gives rise to two distinct proteins, both of about 60 kilodaltons (kDa) (16,18). Little is known about the physiological function of either c-src protein. A number of other proto-oncogene products are involved in the transduction of normal mitogenic signals (e.g., c-erbB [epidermal growth factor receptor], c-fms [colony-stimulating factor 1 receptor], c-sis [platelet-derived growth factor]). However, the tissue distribution of pp6Oc-src expression suggests that this is not its function; those cells which express the highest levels of pp60csrc do not divide. For example, postmitotic differentiated or differentiating neurons express high levels of both c-src proteins (1,3,7,8,17,21). Mammalian platelets, terminally differentiated cells with no growth potential, express still higher levels of the smaller c-src protein (9). These observations suggest that tyrosine-specific protein kinases in general, and pp60c-sr in particular, may regulate cellular processes that are distinct from growth control.We have chosen human platelets as a system for investigating the role of protein-tyrosine phosphorylation in differentiated cells. Platelets are relatively simple cell fragments (they do not syn...

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Micro-scale chromophore-assisted laser inactivation of nerve growth cone proteins

Buchstaller

Jay

2000

Microsc. Res. Tech.

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Directed growth cone movement is crucial for the correct wiring of the nervous system. This movement is governed by the concerted actions of cell surface receptors, signaling proteins, cytoskeleton‐associated molecules, and molecular motors. In order to investigate the molecular basis of growth cone motility, we applied a new technique to functionally inactivate proteins: micro‐scale Chromophore‐Assisted Laser Inactivation [Diamond et al. (1993) Neuron 11:409–421]. Micro‐CALI uses laser light of 620 nm, focused through microscope optics into a 10‐μm spot. The laser energy is targeted via specific Malachite green‐labeled, non‐function‐blocking antibodies, that generate short‐lived protein‐damaging hydroxyl radicals [Liao et al. (1994) Proc Natl Acad Sci USA 91:2659–2663]. Micro‐CALI mediates specific loss of protein function with unachieved spatial and temporal resolution. Combined with time‐lapse video microscopy, it offers the possibility to induce and observe changes in growth cone dynamics on a real time base. We present here the effects of the acute and localized inactivation of selected growth cone molecules on growth cone behavior and morphology. Based on our observations, we propose specific roles for these proteins in growth cone motility and neurite outgrowth. Microsc. Res. Tech. 41:97–106, 2000. © 2000 Wiley‐Liss, Inc.

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Neurones express high levels of a structurally modified, activated form of pp60c-src

Cited by 364 publications

References 31 publications

Synapsin I interacts with c-Src and stimulates its tyrosine kinase activity

Synapsin I interacts with c-Src and stimulates its tyrosine kinase activity

Platelet tyrosine-specific protein phosphorylation is regulated by thrombin.

Micro-scale chromophore-assisted laser inactivation of nerve growth cone proteins

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