The Cbl proto-oncogene product negatively regulates the Src-family tyrosine kinase Fyn by enhancing its degradation.

Fyn is a prototype Src-family tyrosine kinase that plays specific roles in neural development, keratinocyte differentiation, and lymphocyte activation, as well as roles redundant with other Src-family kinases. Similar to other Src-family kinases, efficient regulation of Fyn is achieved through intramolecular binding of its SH3 and SH2 domains to conserved regulatory regions. We have investigated the possibility that the tyrosine kinase regulatory protein Cbl provides a complementary mechanism of Fyn regulation. We show that Cbl overexpression in 293T embryonic kidney and Jurkat T-lymphocyte cells led to a dramatic reduction in the active pool of Fyn; this was seen as a reduction in Fyn autophosphorylation, reduced phosphorylation of in vivo substrates, and inhibition of transcription from a Src-family kinase response element linked to a luciferase reporter. Importantly, a Fyn mutant (FynY528F) relieved of intramolecular repression was still negatively regulated by Cbl. The Cbl-dependent negative regulation of Fyn did not appear to be mediated by inhibition of Fyn kinase activity but was correlated with enhanced protein turnover. Consistent with such a mechanism, elevated levels of Fyn protein were observed in cell lines derived from Cbl(-/-) mice compared to those in wild-type controls. The effects of Cbl on Fyn were not observed when the 70ZCbl mutant protein was analyzed. Taken together, these observations implicate Cbl as a component in the negative regulation of Fyn and potentially other Src-family kinases, especially following kinase activation. These results also suggest that protein degradation may be a general mechanism for Cbl-mediated negative regulation of activated tyrosine kinases.

c-Cbl localizes to actin lamellae and regulates lamellipodia formation and cell morphology.

Adhesive and locomotive properties of cells have key roles in normal physiology and disease. Cell motility and adhesion require the assembly and organization of actin microfilaments into stress fibers, lamellipodia and filopodia, and the formation of these structures is mediated by signalling through Rho; GTPases. Here we identify c-Cbl (a multi-adaptor proto-oncogene product involved in protein tyrosine kinase signalling) as an important regulator of the actin cytoskeleton. By immunofluorescence microscopy we have determined that c-Cbl co-localizes with the adaptor protein Crk to submembranous actin lamellae in NIH 3T3 fibroblasts and that c-Cbl's actin localization requires specific SH3-binding sequences. Further, we have found that truncation of this SH3-binding domain in c-Cbl profoundly alters the morphology of NIH 3T3 fibroblasts by inhibiting the formation of actin lamellae, lamellipodia and membrane ruffles. The induction of lamellipodia and membrane ruffles are also inhibited during cell spreading and migration, conditions when these structures are normally most prominent. The inhibitory effect of truncated c-Cbl expression on lamellipodia formation can be reversed by mutational inactivation of its divergent SH2 domain, by the co-expression of constitutively active Rac or by the overexpression of c-Cbl. This study therefore identifies a cytoskeletal role for c-Cbl which may involve the regulation of Crk and Rac, and which is dependent on targeting of c-Cbl to actin lamellae and the ability to recruit signalling protein(s) associated with its divergent SH2 domain.

Alteration of endothelial cell monolayer integrity triggers resynthesis of vascular endothelium cadherin.

Although cadherins appear to be necessary for proper cell-cell contacts, the physiological role of VE-cadherin (vascular endothelium cadherin) in adult tissue has not been clearly determined. To shed some light on this question, we have disturbed the adhesive function of VE-cadherin in human endothelial cell culture using a polyclonal anti-VE-cadherin antibody. This antibody disrupts confluent endothelial cell monolayers in vitro and transiently generates numerous gaps at cell-cell junctions. The formation of these gaps correlates with a reversible increase in the monolayer permeability. We present evidence that destruction of the homotypic interactions between the extracellular domains of VE-cadherin induces a rapid resynthesis of VE-cadherin, leading to restoration of endothelial cell-cell contacts. The expression of new molecules of VE-cadherin correlates with a modest but significant increase in VE-cadherin mRNA synthesis. Altogether, these results establish a critical role for VE-cadherin in the maintenance and restoration of endothelium integrity.

Dual function C-terminal domain of dynamin-1: modulation of self-assembly by interaction of the assembly site with SH3 domains.

Impairment of endocytosis by mutational targeting of dynamin-1 GTPases can result in paralysis and embryonic lethality. Dynamin-1 assembles at coated pits where it functions to cleave vesicles from donor membranes. Receptor endocytosis is modulated by SH3 (src homology 3) domain proteins, which directly bind to dynamin C-terminal proline motif sequences, affecting both the dynamin GTPase activity and its recruitment to coated pits. We have determined that dynamin-dynamin interactions, which are required for dynamin helix formation, involve these same SH3 domain-binding C-terminal proline motif sequences. Consequently, SH3 domain proteins induce the in vitro disassembly of dynamin helices. Our results therefore suggest the the dual function of the dynamin C-terminus (involving amino acids 800-840) permits direct regulation of dynamin assembly and function through interaction with SH3 domain proteins. Additionally, the N-terminal GTPase domain plays an important role in assembly. Finally, we show that the central PH (pleckstrin homology) domain exerts a strong inhibitory effect on the capacity for dynamin-1 self-assembly.

The role of the PH domain and SH3 binding domains in dynamin function.

Dynamin, a 100 kD GTPase, is necessary for the normal development and function of mammalian neural tissue. In neurons, it is necessary for the biogenesis of synaptic vesicles, and in other cell types dynamin has a general and important role in clathrin mediated receptor endocytosis. Different isoforms function as molecular scissors either during the formation of coated vesicles from plasma membrane coated pits, or during the release of intracellular vesicles from donor membranes. The mechanism entails the formation of a horseshoe-shaped dynamin polymer at the neck of the budding vesicle, followed by neck scission through a GTP hydrolysis dependent activity. The primary sequence of dynamin contains several C-terminal SH3 binding proline motifs, a central pleckstrin homology (PH) domain, and an N-terminal GTPase domain. Each of these domains appears to play a distinct role in dynamin function. Dynamin is activated by stimulus coupled PKC phosphorylation in brain, possibly mediated through PKC interactions with the PH domain. Further, SH3 domain interactions with the C-terminal sequences and phophatidylinositol/G beta gamma interactions with the PH domain also increase dynamin GTPase activity. Each of these various regulatory mechanisms is important in dynamin function during vesicle budding, although the means by which these mechanisms integrate in the overall function of dynamin remains to be elucidated.

Distinct specificity in the recognition of phosphoinositides by the pleckstrin homology domains of dynamin and Bruton's tyrosine kinase.

Pleckstrin homology (PH) domains may act as membrane localization modules through specific interactions with phosphoinositide phospholipids. These interactions could represent responses to second messengers, with scope for regulation by soluble inositol polyphosphates. A biosensor-based assay was used here to probe interactions between PH domains and unilamellar liposomes containing different phospholipids and to demonstrate specificity for distinct phosphoinositides. The dynamin PH domain specifically interacted with liposomes containing phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] and, more weakly, with liposomes containing phosphatidylinositol-4-phosphate [PI(4)P]. This correlates with phosphoinositide activation of the dynamin GTPase. The functional GTPase of a dynamin mutant lacking the PH domain, however, cannot be activated by PI(4,5)P2. The phosphoinositide-PH domain interaction can be abolished selectively by point mutations in the putative binding pocket predicted by molecular modelling and NMR spectroscopy. In contrast, the Bruton's tyrosine kinase (Btk)PH domain specifically bound liposomes containing phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3]: an interaction requiring Arg28, a residue found to be mutated in some X-linked agammaglobulinaemia patients. A rational explanation for these different specificities is proposed through modelling of candidate binding pockets and is supported by NMR spectroscopy.

Two isoforms of Drosophila dynamin in wild-type and shibire(ts) neural tissue: different subcellular localization and association mechanisms.

The temperature-sensitive mutations of the shibire (shi) gene in Drosophila cause endocytic arrest, resulting in neurotransmission block and paralysis at high temperatures. However, underlying mechanism for the defects is not yet known. We examined the subcellular distribution of dynamin, a product of the shi gene, by immunoblotting and immunocytochemical assays. Two isoforms of dynamin with apparent M(r) of 92 kD and 94 kD have been detected in wild-type and shi(n) adult neural tissue. The two isoforms were reproducibly associated with different subcellular fractions of head homogenates. The 94kD isoform is fractionated in the low speed (2.000 x g) pellet containing plasma membrane fragments, and the 92kD isoform in the high speed (130,000 x g) pellet. In this procedure, very little dynamin remained in the high speed supernatant fraction. The 94 kD isoform represents the majority (65-75%) of total dynamin and appears to be a peripheral membrane protein. It can be extracted from the low speed membrane pellet by high salt, Na2CO3 (pH 11) or Triton X-100 treatments. Extracted 94kD dynamin from both wild-type and mutant homogenates is able to reassociate with artificial phospholipid vesicles at both permissive and restrictive temperatures. Binding of the 94 kD dynamin to liposomes appears to be pH-dependent, varying most significantly within the physiological pH range, which may be functionally important. The 92 kD isoform cannot be released by high salt or Na2CO3 treatments and only a small fraction is released by Triton X-100, suggesting a different mechanism of association with cell structures. The distribution of the two isoforms is not altered by the presence of stabilized microtubules in homogenates. No apparent degradation or subcellular redistribution of mutant dynamin was detected in two shi(n) alleles after heat shock or block of the dynamin GTPase activity, suggesting that intracellular redistribution or degradation of mutant dynamin are not involved in the endocytosis arrest in these mutants. These observations resemble the effect of endocytosis arrest by GTP-gamma-S in rat brain synaptosomes (Takei et al., 1995), in which dynamin is trapped at the neck of invaginated pits but is absent in the clathrin-coated distal end undergoing internalization. Our finding that endocytosis arrest by shi(n) mutations and GTP-gamma-S do not lead to cumulation of dynamin in the low speed pellet fraction further suggests that the 94 kD isoform remains associated with the plasma membrane during coated vesicle pinch-off and that the two isoforms do not appear to correspond to different functional states of dynamin but are likely to be involved in separate cellular compartments within the membrane cycling pathway (e.g., the plasma membrane, endosomes, and endoplasmic reticulum).

Growth factor-induced binding of dynamin to signal transduction proteins involves sorting to distinct and separate proline-rich dynamin sequences.

Dynamin, a 100 kDa GTPase, is critical for endocytosis, synaptic transmission and neurogenesis. Endocytosis accompanies receptor processing and plays an essential role in attenuating receptor tyrosine kinase signal transduction. Dynamin has been demonstrated to be involved in the endocytic processing at the cell surface and may play a general role in coupling receptor activation to endocytosis. Src homology (SH) domain dependent protein-protein interactions are important to tyrosine kinase receptor signal transduction. The C-terminus of dynamin contains two clusters of SH3 domain binding proline motifs; these motifs may interact with known SH3 domain proteins during tyrosine kinase receptor activation. We demonstrate here that SH3 domain-containing signal transduction proteins, such as phospholipase C gamma-1 (PLC gamma-1), do indeed bind to dynamin in a growth factor inducible manner. The induction of PLC gamma-1 binding to dynamin occurs within minutes of the addition of platelet derived growth factor (PDGF) to cells. Binding of these signal transduction proteins to dynamin involves specific sorting to individual proline motif clusters and appears to be responsible for co-immunoprecipitation of tyrosine phosphorylated PDGF receptors with dynamin following PDGF stimulation of mammalian cells. The binding of dynamin to SH3 domain-containing proteins may therefore be important for formation of the protein complex required for the endocytic processing of activated tyrosine kinase receptors.

ADPRibosylation of chicken red cell tubulin and inhibition of microtubule self-assembly in vitro by the NAD(+)-dependent avian ADPRibosyl transferase.

Chicken erythrocyte tubulin was found to undergo NAD(+)-dependent ADPribosylation in vitro in the presence of ADPRtransferase also isolated from avian red blood cells. Unlike the low level of ADPR incorporation catalyzed by Cholera and Pertussis toxins (i.e., less than 0.005 mol ADPR/mol tubulin), the avian system displayed a much higher stoichiometry of 0.8-1.2 mol ADPR/mol tubulin. Modification resulted in potent inhibition of microtubule self-assembly, even in the presence of bovine brain microtubule-associated proteins or with the addition of pre-assembled microtubules.

Microtubule protein ADP-ribosylation in vitro leads to assembly inhibition and rapid depolymerization.

Bovine brain microtubule protein, containing both tubulin and microtubule-associated proteins, undergoes ADP-ribosylation in the presence of [14C]NAD+ and a turkey erythrocyte mono-ADP-ribosyltransferase in vitro. The modification reaction could be demonstrated in crude brain tissue extracts where selective ADP-ribosylation of both the alpha and beta chains of tubulin and of the high molecular weight microtubule-associated protein MAP-2 occurred. In experiments with purified microtubule protein, tubulin dimer, the high molecular weight microtubule-associated protein MAP-2, and another high molecular weight mirotubule-associated protein which may be a MAP-1 species were heavily labeled. Tubulin and MAP-2 incorporated [14C]ADP-ribose to an average extent of approximately 2.4 and 30 mol of ADP-ribose/mol of protein, respectively. Assembly of microtubule protein into microtubules in vitro was inhibited by ADP-ribosylation, and incubation of assembled steady-state microtubules with ADP-ribosyltransferase and NAD+ resulted in rapid depolymerization of the microtubules. Thus, the eukaryotic enzyme can ADP-ribosylate tubulin and microtubule-associated proteins to much greater extents than previously observed with cholera and pertussis toxins, and the modification can significantly modulate microtubule assembly and disassembly.

Biochemical and immunochemical analysis of rat brain dynamin interaction with microtubules and organelles in vivo and in vitro.

We have purified a 100-kD rat brain protein that has microtubule cross-linking activity in vitro, and have determined that it is dynamin, a putative microtubule-associated motility protein. We find that dynamin appears to be specific to neuronal tissue where it is present in both soluble and particulate tissue fractions. In the cytosol it is abundant, representing as much as 1.5% of the total extractable protein. Dynamin appears to be in particulate material due to association with a distinct subcellular membrane fraction. Surprisingly, by immunofluorescence analysis of PC12 cells we find that dynamin is distributed uniformly throughout the cytoplasm with no apparent microtubule association in either interphase, mitotic, or taxol-treated cells. Upon nerve growth factor (NGF) induction of PC12 cell differentiation into neurons, dynamin levels increase approximately twofold. In the cell body, the distribution of dynamin again remains clearly distinct from that of tubulin, and in axons, where microtubules are numerous and ordered into bundles, dynamin staining is sparse and punctate. On the other hand, in the most distal domain of growth cones, where there are relatively few microtubules, dynamin is particularly abundant. The dynamin staining of neurites is abolished by extraction of the cells with detergent under conditions that preserve microtubules, suggesting that dynamin in neurites is associated with membranes. We conclude that dynamin is a neuronal protein that is specifically associated with as yet unidentified vesicles. It is possible, but unproven, that it may link vesicles to microtubules for transport in differentiated axons.