Abstract. A phosphorylated epitope is differentially expressed at the kinetochores of chromosomes in mitotic cells and may be involved in regulating chromosome movement and cell cycle progression. During prophase and early prometaphase, the phosphoepitope is expressed equally among all the kinetochores. In mid-prometaphase, some chromosomes show strong labeling on both kinetochores; others exhibit weak or no labeling; while in other chromosomes, one kinetochore is intensely labeled while its sister kinetochore is unlabeled. Chromosomes moving toward the metaphase plate express the phosphoepitope strongly on the leading kinetochore but weakly on the trailing kinetochore. This is the first demonstration of a biochemical difference between the two kinetochores of a single chromosome. During metaphase and anaphase, the kinetochores are unlabeled. At metaphase, a single misaligned chromosome can inhibit further progression into anaphase. Misaligned chromosomes express the phosphoepitope strongly on both kinetochores, even when all the other chromosomes of a cell are assembled at the metaphase plate and lack expression. This phosphoepitope may be involved in regulating chromosome movement to the metaphase plate during prometaphase and may be part of a cell cycle checkpoint by which the onset of anaphase is inhibited until complete metaphase alignment is achieved.

We show that gamma-glutamyl transpeptidase (GGT) is a glutathionase that enables cells to use extracellular glutathione as a source of cysteine. We transfected NIH/3T3 mouse fibroblasts with a plasmid containing cDNA for human GGT, and obtained stably transformed cell lines that expressed GGT in its proper orientation on the outer surface of the cell. NIH/3T3 fibroblasts require cysteine for growth and are unable to use extracellular glutathione as a source of cysteine. We demonstrate GGT-positive fibroblasts are able to grow in cysteine-free medium supplemented with glutathione. Cysteine derived from the cleavage of extracellular glutathione can be used to maintain intracellular levels of glutathione. GGT-positive NIH/3T3 cells were able to replenish intracellular glutathione when incubated in cysteine-free medium containing glutathione. GGT-negative cells could not. Therefore, GGT is a glutathionase that provides the cell with access to a secondary source of cysteine.

We examined the role of heterotrimeric G protein signaling components in insulin and insulin-like growth factor I (IGF-I) action. In HIRcB cells and in 3T3L1 adipocytes, treatment with the G␣ i inhibitor (pertussis toxin) or microinjection of the G␤␥ inhibitor (glutathione S-transferase-␤ARK) inhibited IGF-I and lysophosphatidic acid-stimulated mitogenesis but had no effect on epidermal growth factor (EGF) or insulin action. In basal state, G␣ i and G␤ were associated with the IGF-I receptor (IGF-IR), and after ligand stimulation the association of IGF-IR with G␣ i increased concomitantly with a decrease in G␤ association. No association of G␣ i was found with either the insulin or EGF receptor. Microinjection of anti-␤-arrestin-1 antibody specifically inhibited IGF-I mitogenic action but had no effect on EGF or insulin action. ␤-Arrestin-1 was associated with the receptors for IGF-I, insulin, and EGF in a ligand-dependent manner. We demonstrated that G␣ i , ␤␥ subunits, and ␤-arrestin-1 all play a critical role in IGF-I mitogenic signaling. In contrast, neither metabolic, such as GLUT4 translocation, nor mitogenic signaling by insulin is dependent on these protein components. These results suggest that insulin receptors and IGF-IRs can function as G protein-coupled receptors and engage different G protein partners for downstream signaling.Although the insulin-like growth factor I receptor (IGF-IR) 1 and the insulin receptor (IR) are structurally and functionally related heterotetrameric proteins and share many of the same signaling molecules, they modulate different responses within the cell. IGF-I has been implicated mostly in mitogenic functions and insulin in metabolic actions (1, 2). The insulin and IGF-I receptors consist of two extracellular ␣-subunits and two transmembrane ␤-subunits and are members of the receptor tyrosine kinase (RTK) class of membrane localized receptors (2-4). Ligand binding activates the tyrosine kinase activity of the ␤ subunits (2-4) leading to autophosphorylation, as well as tyrosine phosphorylation of a variety of endogenous substrates, such as the IRS proteins (5), Shc (6, 7), Gab1 (8), and G␣ q/11 (9). It is thought that these endogenous substrates then go on to mediate the biologic effects of these two hormones through a variety of mechanisms (5-9). Heptahelical receptors are another broad class of membrane receptors, and this receptor class is often referred to as G protein-coupled receptors (GPCRs), because they exert their biologic effects by interacting with a family of heterotrimeric G protein signaling molecules (10 -12).Although it is often thought that the RTKs and heptahelical/ GPCRs represent structurally and functionally different classes of receptor types, recent evidence indicates that this may not be strictly the case (13-16). For example, it has now been established that specific heptahelical receptor biologic responses, such as activation of MAP kinase (16 -19), can be mediated by tyrosine kinase events initiated through activation of Src kinase (20 -24). Furt...

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Protein phosphatase 2A (PP2A) is a multimeric serine/threonine phosphatase that carries out multiple functions. Although numerous observations suggest that PP2A plays a major role in downregulation of the mitogen-activated protein (MAP) kinase pathway, the precise mechanisms are unknown. To clarify the role of PP2A in growth factor (insulin, epidermal growth factor [EGF], and insulin-like growth factor 1 [IGF-1]) stimulation of the Ras/MAP kinase pathway, simian virus 40 small t antigen was expressed in Rat-1 fibroblasts which overexpress insulin receptors. Small t antigen is known to specifically inhibit PP2A by binding to the A PP2A regulatory subunit, interfering with the ability of PP2A to bind to its cellular substrates. Overexpressed small t protein was coimmunoprecipitated with PP2A and inhibited cellular PP2A activity but did not inhibit protein phosphatase 1 (PP1) activity. Insulin, IGF-1, and EGF stimulation also inhibited PP2A activity. Growth factor-stimulated Ras, Raf-1, MAP kinase, and mitogen-activated extracellular-signal-regulated kinase kinase (MEK) activities were elevated in small-t-antigen-expressing cells. Furthermore, Shc tyrosine phosphorylation and its association with Grb2 were also elevated in small-t-antigen-expressing cells. Expression levels of Shc, Ras, MEK, or MAP kinase and phosphorylation of insulin, EGF, and IGF-1 receptors were not altered. Interestingly, we found that PP2A associated with Shc in the basal state and dissociated in response to insulin and EGF and that this dissociation was inhibited by 65% in small-t-antigen-expressing cells. In addition, we found that PP2A associates with the phosphotyrosine-binding domain (PTB domain) of Shc and that phosphorylation of tyrosine 317 of Shc was required for PP2A-Shc dissociation. We conclude (i) that PP2A negatively regulates the Ras/MAP kinase pathway by binding to Shc, inhibiting tyrosine phosphorylation; (ii) that the Shc-PP2A association is mediated by the Shc PTB domain but the interaction is independent of phosphotyrosine binding, indicating a new molecular function for the PTB domain; (iii) that growth factor stimulation, or small-t-antigen expression, causes dissociation of the PP2A-Shc complex, facilitating Shc phosphorylation and downstream activations of the Ras/MAP kinase pathway; and (iv) that this defines a new mechanism of small-t-antigen action to promote mitogenesis.Protein phosphorylation plays a key role in many cellular processes, including signal transduction pathways (23, 32). The phosphorylation state of a target protein is regulated by opposing kinases and phosphatases (23). Protein phosphatase 2A (PP2A) is a major cytoplasmic serine/threonine phosphatase that plays an important role in the regulation of cell growth and a diverse set of cellular proteins, including metabolic enzymes, ion channels, hormone receptors, and kinase cascades (14,30,50). It is ubiquitously expressed and accounts for a significant fraction of serine/threonine phosphatase activity in most tissue and cell types (14,30,50). Native ...

Shc is involved in the activation of Ras in response to many growth factors. Shc contains two phosphotyrosine binding domains, an Src homology 2 (SH2) domain in the carboxyl terminus of the protein and a phosphotyrosine binding (PTB) domain in the amino terminus. Since functional roles for these two domains have not been established, we microinjected glutathione S-transferase fusion proteins of either the Shc PTB or SH2 domains into fibroblasts expressing insulin and epidermal growth factor receptors and measured their effects on DNA synthesis. We found that the Shc PTB was necessary for insulin-induced mitogenic signaling, whereas the SH2 domain was not. In contrast, for epidermal growth factor signaling, the Shc SH2 was functionally more important. These differential modes of signal transduction may be an important factor in determining the specificity of the response of a cell to external stimuli.Shc is an Src homology 2 (SH2) 1 domain containing adapter protein that becomes phosphorylated on tyrosine residues in response to many growth factors (1-4). Tyrosine phosphorylation of Shc leads to the activation of p21 ras (5, 6). The functions of Shc in signal transduction are mediated by several domains within the protein. Shc contains two phosphotyrosine binding domains, a proline-rich region referred to as the collagen homology (CH) domain and a major site of tyrosine phosphorylation at tyrosine 317 to which the SH2 domain of Grb2 binds (7). Of importance to this study are the phosphotyrosine binding domains that are located at the opposite ends of the protein. An SH2 domain is located at the carboxyl terminus of the protein and a phosphotyrosine binding (PTB, also known as the PID or SAIN) domain is located near the amino terminus (8 -10).SH2 domains are regions of approximately 100 amino acids that are similar to a region in Src and are able to bind to phosphotyrosines and sequences carboxyl-terminal to the phosphotyrosine (for review, see Refs. 11 and 12). In contrast, the PTB domain is not as clearly defined and recognizes phosphotyrosines and sequences amino-terminal to the phosphotyrosine (13-15), specifically those that form a ␤ turn (16). Although both domains have been shown to bind to many phosphotyrosyl-containing proteins after stimulation of a cell with growth factors, the functional role of the Shc SH2 and PTB have not been established.In this study, we performed single cell microinjection experiments in fibroblasts expressing both the insulin and epidermal growth factor (EGF) receptors in which reagents which inhibited either the endogenous Shc PTB or SH2 domains were introduced into the cell interior. Reagents that disrupted PTB domain interactions of endogenous Shc were shown to inhibit insulin stimulated mitogenesis but had only a small effect on EGF-stimulated mitogenesis. In comparison, reagents that disrupted SH2 interactions of endogenous Shc inhibited only EGF-stimulated cell cycle progression. Therefore, although both the insulin receptor and the EGF receptor activate Ras through Shc, di...

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Multi-drug chemotherapy resistance in non-small cell lung cancer tumor cultures is common, and associations between molecular markers and in vitro chemotherapy resistance are noted. Clinical validation through integration of such testing into clinical trials seems warranted.