David Shalloway

Cell cycle-dependent activation of Ras

BACKGROUND Ras proteins play an essential role in the transduction of signals from a wide range of cell-surface receptors to the nucleus. These signals may promote cellular proliferation or differentiation, depending on the cell background. It is well established that Ras plays an important role in the transduction of mitogenic signals from activated growth-factor receptors, leading to cell-cycle entry. However, important questions remain as to whether Ras controls signalling events during cell-cycle progression and, if so, at which point in the cell-cycle it is activated. RESULTS To address these questions we have developed a novel, functional assay for the detection of cellular activated Ras. Using this assay, we found that Ras was activated in HeLa cells, following release from mitosis, and in NIH 3T3 fibroblasts, following serum-stimulated cell-cycle entry. In each case, peak Ras activation occurred in mid-G1 phase. Ras activation in HeLa cells at mid-G1 phase was dependent on RNA and protein synthesis and was not associated with tyrosine phosphorylation of Shc proteins and their binding to Grb2. Significantly, activation of Ras and the extracellular-signal regulated (ERK) sub-group of mitogen-activated protein kinases were not temporally correlated during G1-phase progression. CONCLUSIONS Activation of Ras during mid-G1 phase appears to differ in many respects from its rapid activation by growth factors, suggesting a novel mechanism of regulation that may be intrinsic to cell-cycle progression. Furthermore, the temporal dissociation between Ras and ERK activation suggests that Ras targets alternate effector pathways during G1-phase progression.

A phosphotyrosine displacement mechanism for activation of Src by PTPα

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

Incorporation of the gene for a cell-cell channel protein into transformed cells leads to normalization of growth.

SummaryIncorporation of the gene for connexin 43, a cell-cell channel protein of gap junction, into the genome of communication-deficient transformed mouse 10T1/2 cells restored junctional communication and inhibited growth. Growth was slowed, saturation density reduced and focus formation suppressed, and these effects were contingent on overexpression of the exogenous gene and the consequent enhancement of communication. In coculture with normal cells the growth of the connexin overexpressors was completely arrested, as these cells established strong communication with the normal ones. Thus, in culture by themselves or in coculture, the connexin overexpressor cells grew like normal cells. These results demonstrate that the cell-cell channel is instrumental in growth control; they are the expected behavior if the channel transmits cytoplasmic growth-regulatory signals.

Specificity and Determinants of Sam68 RNA Binding IMPLICATIONS FOR THE BIOLOGICAL FUNCTION OF K HOMOLOGY DOMAINS

Sam68, a specific target of the Src tyrosine kinase in mitosis, possesses features common to RNA-binding proteins, including a K homology (KH) domain. To elucidate its biological function, we first set out to identify RNA species that bound to Sam68 with high affinity using in vitro selection. From a degenerate 40-mer pool, 15 RNA sequences were selected that bound to Sam68 with K d values of 12–140 nm. The highest affinity RNA sequences (K d ∼12–40 nm) contained a UAAA motif; mutation to UACA abolished binding to Sam68. Binding of the highest affinity ligand, G8-5, was assessed to explore the role of different regions of Sam68 in RNA binding. The KH domain alone did not bind G8-5, but a fragment containing the KH domain and a region of homology within the Sam68 subgroup of KH-containing proteins was sufficient for G8-5 binding. Deletion of the KH domain or mutation of KH domain residues analogous to loss-of-function mutations in the human Fragile X syndrome gene product and the Caenorhabditis elegans tumor suppressor protein Gld-1 abolished G8-5 binding. Our results establish that a KH domain-containing protein can bind RNA with specificity and high affinity and suggest that specific RNA binding is integral to the functions of some regulatory proteins in growth and development.

Association between v-Src and protein kinase C δ in v-Src-transformed fibroblasts

In response to the kinase activity of v-Src there is an increase in the membrane association of the novel protein kinase C (PKC) isoform PKC δ (Zang, Q., Frankel, P., and Foster, D. A. (1995) Cell Growth Differ. 6, 1367–1373). We report here that in v-Src-transformed cells PKC δ co-immunoprecipitates with v-Src and is phosphorylated on tyrosine. The tyrosine-phosphorylated PKC δ had reduced enzymatic activity relative to the non-tyrosine-phosphorylated PKC δ from v-Src-transformed cells. The association between Src and PKC δ was dependent upon both an active Src kinase and membrane association. The association between c-Src Y527F and PKC δ was substantially enhanced by mutating a PKC phosphorylation site at Ser-12 in Src to Ala indicating that PKC δ phosphorylation of Src at Ser-12 destabilizes the interaction, possibly in a negative feedback loop. These data demonstrate that upon recruitment of PKC δ to the membrane in v-Src-transformed cells there is the formation of a Src·PKC δ complex in which PKC δ becomes phosphorylated on tyrosine and down-regulated.

Sam68 exerts separable effects on cell cycle progression and apoptosis

BackgroundThe RNA-binding protein Sam68 has been implicated in a number of cellular processes, including transcription, RNA splicing and export, translation, signal transduction, cell cycle progression and replication of the human immunodeficiency virus and poliovirus. However, the precise impact it has on essential cellular functions remains largely obscure.ResultsIn this report we show that conditional overexpression of Sam68 in fibroblasts results in both cell cycle arrest and apoptosis. Arrest in G1 phase of the cell cycle is associated with decreased levels of cyclins D1 and E RNA and protein, resulting in dramatically reduced Rb phosphorylation. Interestingly, cell cycle arrest does not require the specific RNA binding ability of Sam68. In marked contrast, induction of apoptosis by Sam68 absolutely requires a fully-functional RNA binding domain. Moreover, the anti-cancer agent trichostatin A potentiates Sam68-driven apoptosis.ConclusionsFor the first time we have shown that Sam68, an RNA binding protein with multiple apparent functions, exerts functionally separable effects on cell proliferation and survival, dependent on its ability to bind specifically to RNA. These findings shed new light on the ability of signal transducing RNA binding proteins to influence essential cell function. Moreover, the ability of a class of anti-cancer therapeutics to modulate its ability to promote apoptosis suggests that Sam68 status may impact some cancer treatments.

Two mechanisms activate PTPα during mitosis

We show that, dependent on serine hyperphosphorylation, protein tyrosine phosphatase α (PTPα) is activated by two different mechanisms during mitosis: its specific activity increases and its inhibitory binding to Grb2 decreases. The latter effect probably abates Grb2 inhibition of the phosphotyrosine displacement process that is required specifically for Src dephosphorylation and causes a mitotic increase in transient PTPα‐Src binding. Thus, part of the increased protein tyrosine phosphatase activity may be specific for Src family members. These effects cease along with Src activation when cells exit mitosis. Src is not activated in mitosis in PTPα‐knockout cells, indicating a unique mitotic role for this phosphatase. The activation of PTPα, combined with the effects of mitotic Cdc2‐mediated phosphorylations of Src, quantitatively accounts for the mitotic activation of Src, indicating that PTPα is the membrane‐bound, serine phosphorylation‐activated, protein tyrosine phosphatase that activates Src during mitosis.

Temperature dependent reaction coordinates

Temperature-dependent reaction-coordinates are investigated using Brownian dynamics. A functional of the reaction coordinate, which does not have explicit time dependence, is derived. The path that minimizes the functional is defined as the reaction coordinate. The optimal coordinate varies from the steepest descent path at zero temperature to a straight line connecting “reactants” and “products” at high temperatures. An estimate of the time scale of the process is an output of the optimization. A numerical example is provided and adjustments for the Stratonovich calculus are discussed.

Optimization methods for computing global minima of nonconvex potential energy functions

The minimization of potential energy functions plays an important role in the determination of ground states or stable states of certain classes of molecular clusters and proteins. In this paper we introduce some of the most commonly used potential energy functions and discuss different optimization methods used in the minimization of nonconvex potential energy functions. A very complete bibliography is also given.

The cell cycle and c-Src.

The activity of the proto-oncogene encoded c-Src product is tightly regulated in vivo. In recent years, a model has emerged of how this regulation is achieved. In particular, protein kinases and phosphatases that are potential regulators of c-Src activity in the cell cycle have been identified and characterized.