Insulin potentiates EGFR activation and signaling in fibroblasts.

Insulin is an essential hormone for cell growth and potentiates the mitogenic actions of multiple growth factors, including EGF. While potentiation has been shown to be mediated by the upregulation of the cyclin/CDK system, the upstream mechanisms of such synergy have not been elucidated. Our study has examined whether insulin could mediate synergy by enhancing early signaling events of the EGF receptor (EGFR). Tyrosine phosphorylation at the cell periphery of confluent Swiss 3T3 fibroblasts induced by EGF was potentiated by insulin within 2 min of stimulation. Insulin potentiation of EGF-mediated phosphorylation of the EGFR occurred 2 min after stimulation. EGFR transactivation by insulin was not observed. In addition, downstream mitogenic signaling events including ERK1/2 activation and Elk-1 phosphorylation were enhanced in response to insulin and EGF coadministration. This study shows mitogenic synergy between insulin and EGF can occur at the earliest signaling event, receptor phosphorylation, and independent of transactivation.

The phosphoinositide 3-kinase and p70 S6 kinase regulate long-term potentiation in hippocampal neurons.

The mechanisms by which long-term changes in synaptic efficacy (e.g., long-term potentiation) are maintained are not well understood. There is evidence that reorganization of the neuronal actin cytoskeleton is important for consolidation of long-term potentiation. In non-neuronal cells, phosphoinositide 3-kinase and p70 S6 kinase have been shown to regulate actin polymerization. We have investigated the subcellular localization of these enzymes in cultured hippocampal pyramidal neurons and their possible role in hippocampal long-term potentiation. Immunohistochemical analysis revealed enrichment of both enzymes in the growth cones and filopodia of extending neurites, whereas p70 S6 kinase was also present at the soma. Antibodies to the phosphorylated form of p70 S6 kinase confirmed its activity in these locations. Interestingly, both enzymes displayed strong colocalization with F-actin in discrete regions of developing neurites. In hippocampal slices, the maintenance of long-term potentiation was attenuated by either rapamycin or 2-(4-morpholinyl)-8-phenyl-1(4H)-1-benzopyran-4-one, inhibitors of p70 S6 kinase and phosphoinositide 3-kinase, respectively. Our findings provide evidence for a novel biochemical pathway involving phosphoinositide 3-kinase and p70 S6 kinase that is important for the maintenance of hippocampal long-term potentiation, possibly via regulation of actin dynamics.

Lysophosphatidic acid activates the 70-kDa S6 kinase via the lipoxygenase pathway.

Many hormones are known to activate the 70-kDa S6 kinase (p70(S6K)). The signalling pathways mediating p70(S6K) activation are only partially characterized. We investigate, in this report, the mechanisms by which lysophosphatidic acid (LPA) activates p70(S6K). We observed that p70(S6K) activation was conventional, in that it was sensitive to both rapamycin and PI3 kinase inhibition. p70(S6K) activation appeared to be caused by the activation of several phospholipase pathways. LPA was an effective stimulus of phospholipase C induced intracellular calcium mobilization, which appeared to participate in p70(S6K) activation. Similarly, the effect of LPA on p70(S6K) activity was antagonized by butan-1-ol but not butan-2-ol suggesting the involvement of agonist stimulated phospholipase D activity. Further, antagonism of the phospholipase A(2) and lipoxygenase pathways attenuated p70(S6K) activation indicating a novel mechanism of p70(S6K) regulation. We conclude that in Swiss 3T3 cells LPA coordinates activation of several phospholipases to regulate p70(S6K).

MEK, ERK, and p90RSK are present on mitotic tubulin in Swiss 3T3 cells: a role for the MAP kinase pathway in regulating mitotic exit.

The mitogen-activated protein (MAP) kinase pathway has been implicated in cell cycle control for some time. Several reports have suggested a role for this pathway in growth factor stimulation of DNA synthesis, while other reports have proposed a role in the transition of cells through mitosis. Here, we have examined the potential involvement of the extracellular signal-related kinase (ERK)1/2 MAP kinases, their upstream regulators, and downstream effectors in the regulation of mitosis. Inhibition of MAP kinase/ERK kinase (MEK) activity reduced the serum-stimulated DNA synthesis and proliferation of Swiss 3T3 cells. To study the potential mechanisms of this effect, we examined the subcellular localization of members of the MAP kinase pathway including regulators (MEK1/2), substrates (90-kDa ribosomal S6 kinases (RSKs): RSK1, RSK2 and RSK3), and ERK itself. We show that there is enrichment of ERK, MEK, and the RSK enzymes on both the spindle and midbody tubulin of dividing cells. Inhibition of MEK1/2 activity in cells released from mitotic arrest results in an inability of cells to complete mitosis. This failure to exit mitosis correlated with altered cyclin-dependent kinase (cdk) activities. Thus, the MAP kinase pathway may act to coordinate passage through mitosis in Swiss 3T3 fibroblasts by regulation of cdk activity.

Activation of endogenous thrombin receptors causes clustering and sensitization of epidermal growth factor receptors of swiss 3T3 cells without transactivation.

The G protein-coupled thrombin receptor can induce cellular responses in some systems by transactivating the epidermal growth factor (EGF) receptor. This is in part due to the stimulation of ectoproteases that generate EGF receptor ligands. We show here that this cannot account for the stimulation of proliferation or migration by thrombin of Swiss 3T3 cells. Thrombin has no direct effect on the activation state of the EGF receptor or of its downstream effectors. However, thrombin induces the subcellular clustering of the EGF receptor at filamentous actin-containing structures at the leading edge and actin arcs of migrating cells in association with other signaling molecules, including Shc and phospholipase Cgamma1. In these thrombin-primed cells, the subsequent migratory response to EGF is potentiated. Thrombin did not potentiate the EGF-stimulated EGF receptor phosphorylation. Thus, in Swiss 3T3 cells the G protein-coupled thrombin receptor can potentiate the EGF tyrosine kinase receptor response when activated by EGF, and this appears to be due to the subcellular concentration of the receptor with downstream effectors and not to the overall ability of EGF to induce receptor transphosphorylation. Thus, the EGF receptor subcellular localization which is altered by thrombin appears to be an important determinant of the efficacy of downstream EGF receptor signaling in cell migration.

The flightless I protein colocalizes with actin- and microtubule-based structures in motile Swiss 3T3 fibroblasts: evidence for the involvement of PI 3-kinase and Ras-related small GTPases.

The flightless I protein contains an actin-binding domain with homology to the gelsolin family and is likely to be involved in actin cytoskeletal rearrangements. It has been suggested that this protein is involved in linking the cytoskeletal network with signal transduction pathways. We have developed antibodies directed toward the leucine rich repeat and gelsolin-like domains of the human and mouse homologues of flightless I that specifically recognize expressed and endogenous forms of the protein. We have also constructed a flightless I-enhanced green fluorescent fusion vector and used this to examine the localization of the expressed protein in Swiss 3T3 fibroblasts. The flightless I protein localizes predominantly to the nucleus and translocates to the cytoplasm following serum stimulation. In cells stimulated to migrate, the flightless I protein colocalizes with beta-tubulin- and actin-based structures. Members of the small GTPase family, also implicated in cytoskeletal control, were found to colocalize with flightless I in migrating Swiss 3T3 fibroblasts. LY294002, a specific inhibitor of PI 3-kinase, inhibits the translocation of flightless I to actin-based structures. Our results suggest that PI 3-kinase and the small GTPases, Ras, RhoA and Cdc42 may be part of a common functional pathway involved in Fliih-mediated cytoskeletal regulation. Functionally, we suggest that flightless I may act to prepare actin filaments or provide factors required for cytoskeletal rearrangements necessary for cell migration and/or adhesion.

Nuclear and cytoskeletal translocation and localization of heterotrimeric G-proteins.

Heterotrimeric GTP-binding proteins (G-proteins) are involved in a diverse array of signalling pathways. They are generally thought to be membrane-bound proteins, which disassociate on receptor activation and binding of GTP. A model to explain this has been proposed, which is often described as 'the G-protein cycle'. The 'G-protein cycle' is discussed in the present paper in relation to evidence that now exists regarding the non- membranous localization of G-proteins. Specifically, the experimental evidence demonstrating association of G-proteins with the cytoskeleton and the nucleus, and the mechanisms by which G-proteins translocate to these sites are reviewed. Furthermore, the possible effector pathways and the physiological function of G-proteins at these sites are discussed.

Insulin induces epidermal growth factor (EGF) receptor clustering and potentiates EGF-stimulated DNA synthesis in swiss 3T3 cells: a mechanism for costimulation in mitogenic synergy.

In many cellular systems, activation with more than one ligand can produce a cellular response that is greater than the sum of the individual responses to the ligands. This synergy is sometimes referred to as coactivation. In Swiss 3T3 fibroblasts, activation of the epidermal growth factor (EGF) receptor produces a weak induction of DNA synthesis. Insulin has no stimulatory effect on this response. However, in combination, EGF and insulin synergize to cause a large induction of S phase. The underlying cellular biochemistry of this effect has been examined. The data indicate that phospholipase C activation is a major component of agonist-induced DNA synthesis. In contrast, activation of p70 S6 kinase by single agonists was inversely related to their ability to stimulate DNA synthesis. Therefore, it was examined whether stimulation of Swiss 3T3 cells with insulin causes changes in the subcellular distribution of EGF receptors and phospholipase Cgamma1 that could potentially explain the observed synergy or costimulation. It was found that insulin effectively induced the accumulation of EGF receptors on the actin arc of cells without activation of the EGF receptor. In contrast, EGF, when added for several hours, did not cause accumulation of the EGF receptor at this site. However, both EGF and insulin stimulated the accumulation of phospholipase Cgamma1 at the actin arc, which was coincident with the EGF receptor in the case of insulin- stimulated cells. Therefore, it is suggested that the insulin-induced coclustering of the EGF receptor with phospholipase Cgamma1 at the actin arc may allow for greater efficiency of signal transduction, resulting in the synergy observed for these two hormones in stimulation of DNA synthesis.

The flightless I protein localizes to actin-based structures during embryonic development.

The product of the flightless I gene is predicted to provide a link between molecules of an as yet unidentified signal transduction pathway and the actin cytoskeleton. Previous work has shown that weak and severe mutations of the flightless I locus in Drosophila melanogaster cause disruption in the indirect flight muscles and in embryonic cellularization events, respectively, indicative of a regulatory role for the flightless I protein in cytoskeletal rearrangements. A C-terminal domain within flightless I with significant homology to the gelsolin-like family of actin-binding proteins has been identified, but evidence of a direct interaction between endogenous flightless I and actin remains to be shown. In the present study, chick, mouse and Drosophila melanogaster embryos have been examined and the localization of flightless I investigated in relation to the actin cytoskeleton. It is shown that flightless I localization is coincident with actin-rich regions in parasympathetic neurons harvested from chicks, in mouse blastocysts and in structures associated with cellularization in Drosophila melanogaster.

Cellular function of p70S6K: a role in regulating cell motility.

The 70 kDa ribosomal S6 kinase (p70S6K) is activated by numerous mitogens, growth factors and hormones. Activation of p70S6K occurs through phosphorylation at a number of sites and the primary target of the activated kinase is the 40S ribosomal protein S6, a major component of the machinery involved in protein synthesis in mammalian cells. In addition to its involvement in regulating translation, p70S6K activation has been implicated in cell cycle control and neuronal cell differentiation. Recent data obtained in this laboratory suggests that p70S6K may also function in regulating cell motility, a cellular response that is important in tumour metastases, the immune response and tissue repair. The present paper reviews the regulation and cellular function of p70S6K and proposes a novel function of p70S6K in regulating cell motility.

The GTP-binding protein G(ialpha) translocates to kinetochores and regulates the M-G(1) cell cycle transition of Swiss 3T3 cells.

The receptor-generated signals that are responsible for driving the cell cycle are incompletely characterised in mammalian cells. It is clear, however, that the cellular messenger systems that stimulate DNA synthesis and mitosis are separable. These are interwoven with biochemical checkpoints that ensure that processes, such as chromosomal replication and microtubule attachment to duplicated chromosomes, are complete before the following phase of the cell cycle is initiated. In some cells, activation of DNA synthesis by factors such as LPA and serum has been shown to require the GTP-binding protein G(i). We have found that G(i) plays an additional role in mitosis activated by both 7-transmembrane receptors and tyrosine kinase receptors, and that this involves the translocation of the alpha-subunit of G(i) (G(ialpha)) to the nucleus. Here we show by confocal microscopy that G(ialpha)migrates to the nucleus near the onset of mitosis in serum-activated Swiss 3T3 cells and binds to the kinetochore region of replicated chromosomes. Inhibition of G(i) function with pertussis toxin had no effect on the induction of DNA synthesis by serum, but cell proliferation was inhibited. Flow cytometric analysis showed that this resulted from retardation of the transition through mitosis and into G(1). Additionally, pertussis toxin impaired the activity of p34(cdc2), a cyclin-dependent kinase involved in the transition from M-phase to G(1), but not the S-phase cyclin, cyclin E. These data show that the G-protein G(i) has a key role in the regulation of mitosis in fibroblasts.

An automated fluorescence based assay of neurite formation.

The assessment of neurite-promoting activity of growth factors and neurotrophins in vitro is usually done by the laborious process of counting neuronal processes manually under a standard microscope. This often requires fixation of the cells so that samples can be counted over a period of days when large numbers of cells or samples are assessed. This, therefore, limits the investigator to a single time point for that plate of cells. In an effort to provide an assay that could be used to screen samples quickly to grade overall neurotrophic activity, and to study time-dependent responses easily, an assay is described that quantitates neurite formation based on automated fluorescence detection. Dissociated embryonic mouse dorsal root ganglion neurons were placed in the upper chamber of 1 microm pore size Fluoroblok (Falcon) transwell chambers. The porous membrane of these transwells is optically opaque. NGF placed in the lower chamber of the transwell induced the production of neurites that preferentially passed from the neuronal cell body in the upper chamber through the pores into the lower NGF-containing chamber. The degree of neurite production was assessed both in live cells and in fixed cells by fluorescent labeling of the neurons and neuronal processes. Quantitation of neurite formation was done with a multiwell fluorescence plate reader and verified by confocal microscopy. This method allows the screening and quantitation of neurotrophic activity in both primary neurons and neuronal cell lines. In addition, this also provides experimental access to neuronal processes that are optically separated from the cell body. The latter aspect may also be of great use where fluorescence measurements within neurites and growth cones, such as for Ca2+ release studies, need to be isolated from contaminating fluorescence or synaptic influences of cell bodies.

Nitric oxide donors selectively potentiate thrombin-stimulated p70(S6k) activity and morphological changes in Swiss 3T3 cells.

Thrombin stimulates both DNA synthesis and cell morphological changes in Swiss 3T3 cells, although the mechanism of signal coordination leading to these responses is unknown. We report here that nitric oxide (NO) donors selectively enhance thrombin-stimulated p70(S6k) activity by 40-60%, an effect that was sustained for 24 h. Potentiation of p70(S6k) also was observed with cGMP analogues indicating that this effect is mediated by cGMP-activated protein kinase. NO donors also induced morphological changes characterized by spindle-shaped cells in confluent, nondividing cells or by extended protrusions from the trailing edge in subconfluent, polarized cells. NO donors had no significant effects on intracellular Ca(2+) mobilization, DNA synthesis, proliferation, or ERKs 1 and 2 and p90RSK activities, indicating that mitogenic responses and cell division are not altered by NO donors. We conclude that NO donors modulate the morphological changes associated with cellular motility in response to thrombin stimulation through selective enhancement of p70(S6k) activity.

A p85 subunit-independent p110alpha PI 3-kinase colocalizes with p70 S6 kinase on actin stress fibers and regulates thrombin-stimulated stress fiber formation in swiss 3T3 cells.

The signaling pathways linking receptor activation to actin stress fiber rearrangements during growth factor-induced cell shape change are still to be determined. Recently our laboratory demonstrated the involvement of p70 S6 kinase (p70(s6k)) activation in thrombin-induced stress fiber formation in Swiss 3T3 cells. The present work shows that thrombin-induced p70(s6k) activation is inhibited by the PI 3-kinase inhibitors wortmannin and LY-294002. These inhibitors also significantly reduced thrombin-induced stress fiber formation, demonstrating a role for PI 3-kinase activity in this process, most likely upstream of p70(s6k). Furthermore, the p110alpha form of PI 3-kinase was localized to actin stress fibers, as was previously shown for p70(s6k), as well as to a golgi-like distribution. In contrast, PI 3-kinase p110gamma colocalized with microtubules. The PI 3-kinase p85 subunit, known to be capable of association with p110alpha, was present in a predominantly golgi-like distribution with no presence on actin filaments, suggesting the existence of distinctly localized PI 3-kinase pools. Immunodepletion of p85 from cell lysates resulted in only partial depletion of p110alpha and p110alpha-associated PI 3-kinase activity, confirming the presence of a p85-free p110alpha pool located on the actin stress fibers. Our data, therefore, point to the importance of subcellular localization of PI 3-kinase in signal transduction and to a novel action of p85 subunit-independent PI 3-kinase p110alpha in the stimulation by thrombin of p70(s6k) activation and actin stress fiber formation.

Competition by second messenger systems for receptor interaction and activation: implications for tissue-specific responses and disease therapy.

1. At any one instant, most receptors are now recognized to be able to stimulate multiple signal transduction pathways in a cell when activated by an appropriate hormone. These different signalling pathways appear to allow for distinct cellular responses, such as cell proliferation, differentiation, and shape change. 2. In addition, many different types of cell will possess the same type of receptor. Therefore, for a hormone to be able to transmit differential signals to the various cell types able to respond to it, cells must discriminate the stimulus at some point. Such discrimination would seem to be an absolute requirement to allow a tissue-specific response to an identical initial stimulus. In theory, this specificity could occur at many points in the receptor signal transduction cascade, including cytosolic receptor coupling systems and tissue/cell-specific responsive genes. 3. The present paper summarizes our work and that of others which has determined some of the coupling systems of G-protein-coupled receptors and tyrosine kinase receptors and how these systems may be interacting. 4. In addition to these theoretical considerations, a potential therapeutic strategy underlies the ability of receptors to couple to more than one signal transduction system. If a response to a hormone were, for example, either cell proliferation or cell morphological change or differentiation and separate receptor-coupled signalling systems were responsible for these effects, pharmacological intervention may allow the transfer from one signalling system to another. If such a change allowed a permanent change to the differentiated phenotype, this could potentially form the basis of a signal-based cancer therapy.

Regulation of thrombin-induced stress fibre formation in Swiss 3T3 cells by the 70-kDa S6 kinase.

The signal transduction systems that mediate growth factor receptor-induced cellular shape change have not been fully elucidated, but are known to involve alterations in the state of actin filaments, termed stress fibres. It now appears from several studies that the GTP-binding protein, Rho, is involved. However, the mechanisms by which Rho is activated, and what effectors Rho in turn stimulates are largely matters of conjecture. The present work shows that thrombin is an effective stimulant of stress fibre formation in Swiss 3T3 cells. In addition, we show the 70 kDa form of S6 kinase (p70s6k) to colocalise with stress fibres in both unstimulated and thrombin-activated cells. Coincident with the thrombin-induced formation of stress fibres is the elevated association p70s6k with the fibres. Pretreatment of cells with rapamycin, to inhibit p70s6k activation, inhibits thrombin-induced stress fibre formation and the associated presence of p70s6k on the fibres, supporting a role for p70s6k in thrombin-stimulated stress fibre formation. Thrombin is also shown to stimulate p70s6k activity and that this is inhibited by rapamycin. Thus, the data presented show that thrombin activates stress fibre formation through stimulation of p70s6k via a non-Gi pathway.

The G-protein G(i) regulates mitosis but not DNA synthesis in growth factor-activated fibroblasts: a role for the nuclear translocation of G(i).

GTP binding proteins, heterotrimeric molecules composed of alpha-, beta-, and gamma-subunits, are known to serve as transducers of information from seven-transmembrane receptors. Activation of G-proteins has been generally considered to involve subunit dissociation, with G(alpha) separating from G(betagamma). However, we have found a receptor activation of G(i) in proliferating cells that differs from these models and involves the subcellular translocation of the alpha-subunit from the cell periphery to the nucleus where G(i alpha) binds to chromatin for the duration of mitosis. This report describes the mechanism of G(i) activation in Swiss 3T3 cells in response to serum, thrombin, and epidermal growth factor, and describes a role for G(i2) in the cell cycle. Agonists were found to be unable to induce the physical dissociation of G(i2) subunits. The alpha- and beta-subunits of G(i2) could be coimmunoprecipitated with a G(i alpha) antibody from both the membrane and nuclear fractions of long-term activated cultures, showing that G(i alpha 2) and G(i beta) are induced to comigrate to the nucleus in response to growth factor receptor activation. G(i2) appears to be activated in part by a postreceptor signal that can be mimicked by protein kinase C activation; this signal may be responsible for the convergence of the signaling mechanisms of these distinct seven-transmembrane and tyrosine kinase receptors. We suggest that translocation of G(i alpha) to the nucleus induced by either thrombin or EGF may occur without subunit dissociation. Functional studies of the role of G(i) showed that pertussis toxin does not block DNA synthesis in Swiss 3T3 fibroblasts induced by serum or thrombin, but that cell proliferation is retarded to each. These results provide direct evidence for a novel mechanism of GTP binding protein activation and for an essential role of G(i) in the induction of cell division by a variety of growth factor receptors. G(i) can carry out this role in control of cellular proliferation through its translocation to the nucleus of mitotic cells.

Signal transduction from membrane to nucleus: the special case for neurons.

Neurons have a unique problem with signal transduction from the membrane in the region of their terminals back to the cell body and nucleus. This distance may be several meters in some nerves in some species, so there is a requirement for some mechanism to stabilize the signal. This review examines two complementary mechanisms for this signal transduction, either by the retrograde axonal transport of the neurotrophic factor together with its receptor, or the transport of a stable activated second messenger molecule. Extrapolation of studies on the fibroblast signal transduction pathway, where it has been shown that G1 can translocate from the membrane to the nucleus, has led to the demonstration of the retrograde axonal transport of several putative signaling molecules. The alpha subunits of both G1 and Gz are retrogradely transported and Gz alpha or possibly the intact heterotrimeric Gz subsequently accumulates in dorsal root ganglia nuclei. Thus Gz1 Gi1 and potentially other G-proteins and distinct signaling molecules may provide additional signal transduction pathways to that of the neurotrophins from terminal to nucleus.

Physical association of Gi2alpha with interleukin-8 receptors.

Interleukin-8 (IL-8), one of the major mediators of the inflammatory response, belongs to a family of chemokines that includes NAP-2 (neutrophil-activating peptide-2) and Gro-alpha and whose biological activities are directed to a great extent toward neutrophils. Two distinct receptors have been described with overlapping, but not identical, binding affinities for IL-8, NAP-2, and Gro-alpha. This study was designed to examine the intracellular pathways activated upon the occupation of each of the IL-8 receptors (IL-8R). The formation of a physical coupling between IL-8 receptors and the alpha-subunit of heterotrimeric G proteins was tested in neutrophils by examining the presence of the former in anti-Galpha immune precipitates. The addition of IL-8 to a suspension of human neutrophils led to a time-dependent detection of IL-8 in anti-Gi2alpha (raised against amino acids 159-168 (LERIAQSDYI) of Gi2alpha) and anti-Gtalpha (raised against the COOH-terminal 10 amino acids (KENLKDCGLF) of Gtalpha), but not anti-Gq, immunoprecipitates. Similar results were obtained in human 293 cells stably transfected with IL-8RA or IL-8RB. The peptide derived from the COOH-terminal sequence of Gt inhibited the co-immunoprecipitation of IL-8R and Gi observed in response to the anti-Gtalpha and anti-Gi2alpha antibodies. On the other hand, the Gi2alpha peptide only inhibited the immunoprecipitation induced by the anti-Gi2alpha antibody. Peptides derived from Gi1alpha or Gi3alpha had no effect in this assay. The introduction of the anti-Gi2alpha or anti-Gtalpha antibodies or their neutralizing peptides, but not the Gi1alpha or Gi3alpha peptides, into 293 IL-8RA or 293 IL-8RB cells completely blocked the calcium responses obtained upon stimulation with IL-8. These results demonstrate that the occupation of either type of IL-8 receptor leads to a physical coupling to the alpha-subunit of Gi2. In addition, the use of the subunit-specific peptides identified two functionally important but distinct regions of Gialpha, one involved in receptor/Gialpha interaction (KENLKDCGLF) and the other mediating downstream signal transmission (LERIAQSDYI). Finally, the results of this study also validate the use of the transfected 293 cell line as a model for the study of the signal transduction pathway(s) initiated by IL-8.

Interaction of the GTP-binding protein Gi2 with a protein kinase A-like kinase in mouse fibroblasts.

We have previously shown that the GTP-binding protein, Gi2 of mouse Balb/c3T3 cells is linked to a serine kinase which phosphorylates the alpha-subunit of Gi itself. In this report we show that Gi is coupled to a second protein kinase. This kinase does not phosphorylate G but phosphorylates another protein bound non-covalently to G. Phosphorylation of the Gi-linked protein induces its release from Gi. Kinase activity is slightly enhanced by GTPyS, suggesting that this kinase may be physiologically regulated by Gi. In an attempt to identify the kinase we have examined the effect of peptide substrates and inhibitors on kinase activity. We found that the protein kinase A inhibitory peptide, PK1 5-24, inhibited the kinase activity, but at concentrations above those usually required to block protein kinase A. The protein kinase A substrate peptide, kemptide, acted as a substrate of the kinase, and was an inhibitor of the phosphorylation of the Gi-linked protein. However, a protein kinase A, catalytic subunit antibody failed to react with any proteins linked to Gi., A protein kinase C inhibitory peptide had no effect on phosphorylation of the Gi-linked protein. Thus, the identity of this kinase has not been resolved, but it may form part of the signalling system of activated Gi in fibroblasts.