Chapter Seven - When Phosphorylation Encounters Ubiquitination: A Balanced Perspective on IGF-1R Signaling.

Cell-surface receptors govern the critical information passage from outside to inside the cell and hence control important cellular decisions such as survival, growth, and differentiation. These receptors, structurally grouped into different families, utilize common intracellular signaling-proteins and pathways, yet promote divergent biological consequences. In rapid processing of extracellular signals to biological outcomes, posttranslational modifications offer a repertoire of protein processing options. Protein ubiquitination was originally identified as a signal for protein degradation through the proteasome system. It is now becoming increasingly recognized that both ubiquitin and ubiquitin-like proteins, all evolved from a common ubiquitin structural superfold, are used extensively by the cell and encompass signal tags for many different cellular fates. In this chapter we examine the current understanding of the ubiquitin regulation surrounding the insulin-like growth factor and insulin signaling systems, major members of the larger family of receptor tyrosine kinases (RTKs) and key regulators of fundamental physiological and pathological states.

PKCη promotes a proliferation to differentiation switch in keratinocytes via upregulation of p27Kip1 mRNA through suppression of JNK/c-Jun signaling under stress conditions.

To maintain epidermal homeostasis, the balance between keratinocyte proliferation and differentiation is tightly controlled. However, the molecular mechanisms underlying this balance remain unclear. In 3D organotypic coculture with mouse keratinocytes and fibroblasts, the thickness of stratified cell layers was prolonged, and growth arrest and terminal differentiation were delayed when PKCη-null keratinocytes were used. Re-expression of PKCη in PKCη-null keratinocytes restored stratified cell layer thickness, growth arrest and terminal differentiation. We show that in 3D cocultured PKCη-null keratinocytes, p27(Kip1) mRNA was downregulated, whereas JNK/c-Jun signaling was enhanced. Furthermore, inhibition of JNK/c-Jun signaling in PKCη-null keratinocytes led to upregulation of p27(Kip1) mRNA, and to thinner stratified cell layers. Collectively, our findings indicate that PKCη upregulates p27(Kip1) mRNA through suppression of JNK/c-Jun signaling. This results in promoting a proliferation to differentiation switch in keratinocytes.

Long-term hormonal regulation of the cAMP-specific phosphodiesterases in cultured FRTL-5 thyroid cells.

Thyrotropin (TSH) and pharmacological agents that elevate intracellular cAMP concentrations potentiate the mitogenic response of FRTL-5 thyroid cells to insulin-like growth factor-I (IGF-I). This study was undertaken to determine the role of cAMP phosphodiesterases (PDEs) in this TSH-dependent regulation. Incubation of FRTL-5 cells with TSH, forskolin, or dibutyryl cAMP gradually induced the PDE activity, and treatment for 24 h produced a marked increase in type 4 high affinity cAMP PDEs. Under basal conditions, transcripts corresponding to PDE4A, PDE4B, PDE4C, and PDE4D were present. Stimulation for 24 h by TSH, forskolin or dibutyryl cAMP induced an increase in mRNA levels of PDE4B, PDE4D, and PDE4C. To understand the role of this cAMP-dependent PDE regulation in the potentiation of the mitogenic response to IGF-I, thymidine incorporation into DNA in response to IGF-I and TSH was measured in the absence or presence of PDE inhibitors. Exposure of the cells to 3-isobutyl-1-methylxanthine (IBMX) or RO 20-1724 had opposing effects on thymidine incorporation into DNA, depending on the stimulus applied. When IGF-I was used alone, both IBMX and RO 20-1724 potentiated IGF-I-stimulated thymidine incorporation. However, when IGF-I and TSH at high concentrations were used in combination, these PDE inhibitors blocked thymidine incorporation into DNA. In addition, these inhibitors depressed the synergistic increase in cyclin D1 and cyclin D- or cyclin E-associated cyclin-dependent kinase (CDK) activity that is induced by TSH and IGF-I. Increased CDK activities have been shown to play a crucial role in progression through the G(1)/S phase of the cell cycle. These data demonstrate that TSH produces marked changes in the cAMP degradative pathway of FRTL-5 cells by regulating the expression of cAMP PDEs. The regulation of the intracellular cAMP levels by this mechanism may contribute to the TSH- and IGF-I-dependent control of the entry into the S phase of the cell cycle through changes in the cyclin/CDK system in FRTL-5 cells.

Tyrosine kinase and phosphatidylinositol 3-kinase activation are required for cyclic adenosine 3',5'-monophosphate-dependent potentiation of deoxyribonucleic acid synthesis induced by insulin-like growth factor-I in FRTL-5 cells.

In previous studies, we showed that pretreatment of rat FRTL-5 thyroid cells with TSH, or other agents that increased intracellular cAMP, markedly potentiated DNA synthesis in response to insulin-like growth factor-I (IGF-I). In addition, we found that TSH pretreatment caused an increase in tyrosine phosphorylation of intracellular proteins including an unidentified 125-kDa protein that was well correlated with the TSH-potentiating effect on DNA synthesis induced by IGF-I. These results suggested that cAMP amplified IGF-I-dependent signals for cell growth through changes of cAMP-dependent tyrosine phosphorylation. The present studies were undertaken to determine how tyrosine kinase activation followed by an increase in tyrosine phosphorylation is required for cAMP-dependent potentiation of DNA synthesis induced by IGF-I in this cell line. First of all, we measured tyrosine kinase or protein-tyrosine phosphatase activities in the cell lysates by the in vitro assay. Chronic treatment with TSH or (Bu)2-cAMP stimulated tyrosine kinase activity in the particulate fraction and protein-tyrosine phosphatase activity in the soluble fraction, suggesting that tyrosine kinase plays more important roles for a cAMP-dependent increase in tyrosine phosphorylation of intracellular proteins. The increased tyrosine kinase activity was sensitive to genistein, a potent tyrosine kinase inhibitor. Genistein abolished both the cAMP-dependent increase in tyrosine phosphorylation of the 125-kDa protein and the enhanced DNA synthesis induced by IGF-I in a similar concentration-dependent manner. The only tyrosine-phosphorylated protein associated with the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase in response to cAMP was 125 kDa. In addition, we found that PI 3-kinase activity bound to p85 subunit significantly increased after (Bu)2cAMP treatment. These results suggested that cAMP stimulates PI 3-kinase through tyrosine phosphorylation of the 125-kDa protein. We then measured DNA synthesis in cells pretreated for 24 h with TSH or (Bu)2cAMP in the absence or presence of LY294002, a PI 3-kinase inhibitor, followed by treatment with IGF-I for 24 h. Presence of LY294002 during TSH or (Bu)2cAMP pretreatment completely abolished cAMP-dependent potentiation of DNA synthesis induced by IGF-I. These results suggest that in FRTL-5 cells cAMP activates genistein-sensitive tyrosine kinases that in turn activate PI 3-kinase activity. These mechanisms appear to be necessary for cAMP-dependent potentiation of the DNA synthesis induced by IGF-I.

Signalling pathways of insulin-like growth factor-I that are augmented by cAMP in FRTL-5 cells.

We have reported that pretreatment of rat FRTL-5 thyroid cells with thyrotropin (TSH) markedly potentiates the mitogenic response to insulin-like growth factor-I (IGF-I). The present study was undertaken to determine whether the augmentation by cAMP of IGF-I-dependent tyrosine phosphorylation of known IGF-I receptor substrates plays an important role in the cAMP-dependent potentiation of DNA synthesis induced by IGF-I. Pretreatment with TSH or dibutyryl cAMP did not affect the IGF-I-dependent tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1). In contrast, cAMP pretreatment potentiated the tyrosine phosphorylation of IRS-2 induced by IGF-I, but did not affect the amount of IRS-2. We found that the IGF-I-dependent tyrosine phosphorylation of 66 kDa Shc (Src homology collagen) was markedly increased by cAMP pretreatment, and that this change was mainly due to an increase in the levels of 66 kDa Shc protein. Under these conditions, cAMP pretreatment significantly increased binding of Grb2 (growth-factor-receptor-bound protein 2) to Shc in response to IGF-I, and activation of MAP kinase (mitogen-activated protein kinase) induced by IGF-I was also enhanced by cAMP. The presence of PD98059, an inhibitor of MEK (MAP-kinase/Erk kinase), during treatment with IGF-I partially inhibited the cAMP-dependent augmentation of DNA synthesis in response to IGF-I. On the other hand, cAMP pretreatment increased binding of the phosphoinositide 3-kinase (PI 3-kinase) p85 subunit to IRS-2, which was reflected in PI 3-kinase activity. LY294002, a PI 3-kinase inhibitor, strongly depressed IGF-I-dependent DNA synthesis after pretreatment with and without TSH or dibutyryl cAMP. Our results suggest that the interaction between cAMP-dependent and IGF-I-dependent pathways leads to an augmentation of cell proliferation, which is mediated, at least in part, through the MAP kinase and PI 3-kinase signalling pathways. These effects are mediated by changes in tyrosine phosphorylation of IGF-I receptor substrates, including IRS-2 and Shc.

Insulin-like growth factor-I-dependent signal transduction pathways leading to the induction of cell growth and differentiation of human neuroblastoma cell line SH-SY5Y: the roles of MAP kinase pathway and PI 3-kinase pathway.

IGF-I regulates cell growth, differentiation, and survival in many cultured nerve cell lines. The present study was undertaken in the human neuroblastoma cell line, SH-SY5Y, to elucidate whether there are differences in the IGF-dependent signal transduction pathways that stimulate proliferation compared to those that induce differentiation. Quiescent SH-SY5Y cells were treated with IGF-I in the presence or absence of PD98059 (an inhibitor of MEK, a MAP kinase kinase) or LY294002 (an inhibitor of PI 3-kinase). Cell growth was assessed by measuring [3H]thymidine incorporation into DNA and cell number. Cell differentiation was assessed by measuring mRNA levels of NPY and neurite outgrowth. IGF-I both induced cell proliferation and differentiation. It stimulated tyrosine phosphorylation of the type I IGF receptor (IGF-IR) beta-subunit, IRS-I, IRS-2, and Shc, and these changes were associated with activation of Erk and Akt. PD98059 inhibited activation of Erk and LY294002 repressed activation of Akt in response to IGF-I, but did not affect tyrosine phosphorylation of the IGF-IR, IRS-1, IRS-2, or Shc. Each PD98059 and LY294002 inhibited IGF-I-dependent cell proliferation in a concentration-dependent manner. In contrast, each of these inhibitors only partially depressed NPY gene expression induced by IGF-I and slightly inhibited IGF-I-mediated neurite outgrowth; however, when both PD98059 and LY294002 were present, IGF-I-dependent NPY gene expression and neurite outgrowth were abolished completely. These results suggest that in these nerve cells, 1) the IGF-I signals through the MAP kinase pathway and PI-3 kinase pathway are independently essential to induce IGF-I-dependent growth, and 2) alternate activation of the MAP kinase pathway and PI 3-kinase pathway is sufficient for the cells to undergo IGF-I-dependent differentiation.

Insulin-like growth factor-I potentiates protein synthesis induced by thyrotropin in FRTL-5 cells: comparison of induction of protein synthesis and DNA synthesis.

In FRTL-5 cells, we and others have shown that TSH and insulin-like growth factor-I (IGF-I) stimulate DNA synthesis synergistically. The present study was undertaken to determine whether interaction between TSH and IGF-I also affects protein synthesis in this cell line, and if so by what mechanism. Quiescent cells were treated with TSH and/or IGF-I and [3H]valine incorporation into the acid-insoluble fraction was measured as a parameter of protein synthesis. Similar to their effects on cell proliferation, TSH or IGF-I alone induced protein synthesis only slightly, but treatment with a combination of TSH and IGF-I (or insulin with about a 100-fold higher concentration than IGF-I) greatly increased protein synthesis. The presence of IGF-I potentiated a TSH-concentration-dependent increase in protein synthesis and in DNA synthesis. In addition, we observed this potentiation when the cells were treated with other cAMP-generating agents and cAMP analogues instead of TSH. We have shown that priming with TSH potentiated DNA synthesis induced by IGF-I, whereas pretreatment with IGF-I enhanced protein synthesis induced by TSH. This observation suggested that protein synthesis and DNA synthesis were potentiated through different mechanisms. From an analysis of cAMP production, it appears that the potentiation of protein synthesis may be explained by an IGF-I-dependent increase in cAMP production induced by TSH at least in part. On the other hand, IGF-I and TSH stimulated (alpha-aminoisobutyric acid (AIB) uptake synergistically, but RNA synthesis induced by IGF-I was depressed by TSH. From these results, we concluded that in FRTL-5 cells, IGF-I potentiated protein synthesis induced by TSH by means of complex mechanisms and the interaction between IGF-I and cAMP-dependent pathways may also have a physiological meaning in regulating protein anabolism.

Interaction between cAMP-dependent and insulin-dependent signal pathways in tyrosine phosphorylation in primary cultures of rat hepatocytes.

The present studies were undertaken to determine whether the interaction between cAMP-dependent and insulin-dependent pathways in primary cultures of rat hepatocytes affects biological functions and tyrosine phosphorylation. Quiescent hepatocytes were pretreated with dibutyryl cAMP or cAMP-generating agents such as glucagon, and then treated or not with insulin. Preincubation for 6 h with dibutyryl cAMP or glucagon enhanced the effect of insulin on DNA synthesis, but not the effect of insulin on amino acid transport or glycogen and protein synthesis. Tyrosine phosphorylation of intracellular proteins was determined by immunoblot analysis using an anti-phosphotyrosine antibody. Maximum tyrosine phosphorylation of a 195 kDa protein, which may be a substrate of insulin receptor kinase, of 175-180 kDa proteins, including insulin receptor substrate (IRS)-1, and of 90-95 kDa proteins, including the insulin receptor beta-subunit, was reached within 30 s of incubation with insulin. Pretreatment for about 3 h with dibutyryl cAMP or cAMP-generating agents clearly increased insulin-dependent tyrosine phosphorylation of the 195 kDa protein, but not IRS-1, IRS-2 or the insulin receptor beta-subunit. Because dibutyryl cAMP and cAMP-generating agents did not increase insulin receptor number or its kinase activity, the effect of cAMP on this potentiation of tyrosine phosphorylation is assumed to be exerted at a step distal to insulin receptor kinase activation. The potentiation by cAMP pretreatment of insulin-stimulated tyrosine phosphorylation may in part be secondary to inhibition of phosphotyrosine phosphatase activity, because cAMP pretreatment blunted the effect of Na3VO4 on the net tyrosine phosphorylation of the 195 kDa protein as compared with cells pretreated with no additive. In summary, the interactions between cAMP-dependent and insulin-dependent pathways that lead to augmentation of DNA synthesis appear to parallel the changes in tyrosine phosphorylation. Further studies will be required to determine whether there is a causal relationship between these phenomena.

The Schizosaccharomyces pombe mra1 gene, which is required for cell growth and mating, can suppress the mating inefficiency caused by a deficit in the Ras1 activity.

BACKGROUND: Schizosaccharomyces pombe Ras1 regulates two downstream pathways, namely the Byr2/Byr1/Spk1 mitogen-activated protein kinase cascade and the Cdc42sp small G protein pathway. The former is relevant to mating and sporulation, whereas the latter is relevant to mating, cell growth and cell morphology. We addressed whether Ras1 has any additional role in the regulation of cell physiology. RESULTS: Using a specific mutation in the effector region of Ras1, we isolated a high-copy-number suppressor of the mating deficiency caused by a decrease of the Ras1 activity. The isolated gene, named mra1, encodes a novel protein of 359 amino acids, which has apparent homologues in rice and budding yeast. Disruption of mra1 indicated that it is essential for cell growth, and mutational analysis indicated that it is required for the promotion of mating. These two functions could be separated by mutations, suggesting that Mra1 is bifunctional. Overexpression of mra1 could also suppress the mating inefficiency caused by either overexpression of gap1, which is a downregulator of Ras1, or loss of function of zfs1, which is a gene relevant to the mating pheromone signalling. However, it could not suppress null mutations in genes involved in the two known pathways downstream of Ras1. CONCLUSIONS: Mra1 is an apparent downstream factor of Ras1, which is essential for cell growth and relevant to mating but is not involved in the maintenance of cell morphology. Mra1 is unlikely to interact directly with the known pathways downstream of Ras1, implying that it may be a factor constituting a third pathway regulated by Ras1.