The serine/threonine protein kinase B (PKB/Akt) is involved in insulin signaling, cellular survival, and transformation. Carboxyl-terminal modulator protein (CTMP) has been identified as a novel PKB binding partner in a yeast two-hybrid screen, and appears to be a negative PKB regulator with tumor suppressor-like properties. In the present study we investigate novel mechanisms by which CTMP plays a role in apoptosis process.
CTMP is localized to mitochondria. Furthermore, CTMP becomes phosphorylated following the treatment of cells with pervanadate, an insulin-mimetic. Two serine residues (Ser37 and Ser38) were identified as novel in vivo phosphorylation sites of CTMP. Association of CTMP and heat shock protein 70 (Hsp70) inhibits the formation of complexes containing apoptotic protease activating factor 1 and Hsp70. Overexpression of CTMP increased the sensitivity of cells to apoptosis, most likely due to the inhibition of Hsp70 function.
Our data suggest that phosphorylation on Ser37/Ser38 of CTMP is important for the prevention of mitochondrial localization of CTMP, eventually leading to cell death by binding to Hsp70. In addition to its role in PKB inhibition, CTMP may therefore play a key role in mitochondria-mediated apoptosis by localizing to mitochondria.
The function of insulin receptor substrate-1 (IRS-1) is regulated by both tyrosine and serine/threonine phosphorylation. Phosphorylation of some serine/threonine residues in IRS-1 dampens insulin signaling, whereas phosphorylation of other serine/threonine residues enhances insulin signaling. Phosphorylation of human IRS-1 at Ser629 was increased by insulin in Chinese hamster ovary cells expressing the insulin receptor (1.26 ± 0.09-fold; P < 0.05) and L6 cells (1.35 ± 0.29-fold; P < 0.05) expressing human IRS-1. Sequence analysis surrounding Ser629 revealed conformity to the consensus phosphorylation sequence recognized by Akt. Phosphorylation of IRS-1 at Ser629 in cells was decreased upon treatment with either an Akt inhibitor or by coexpression with kinase dead Akt, whereas Ser629 phosphorylation was increased by coexpression with constitutively active Akt. In addition, Ser629 of IRS-1 is directly phosphorylated by Akt in vitro. In cells, preventing phosphorylation of Ser629 by a Ser629Ala mutation resulted in increased phosphorylation of Ser636, a known negative regulator of IRS-1, without affecting phosphorylation of Tyr632 or Ser616. Cells expressing the Ser629Ala mutation, along with increased Ser636 phosphorylation, had decreased insulin-stimulated association of the p85 regulatory subunit of phosphatidylinositol 3′-kinase with IRS-1 and decreased phosphorylation of Akt at Ser473. Finally, in vitro phosphorylation of a Ser629-containing IRS-1 fragment with Akt reduces the subsequent ability of ERK to phosphorylate Ser636/639. These results suggest that a feed-forward mechanism may exist whereby insulin activation of Akt leads to phosphorylation of IRS-1 at Ser629, resulting in decreased phosphorylation of IRS-1 at Ser636 and enhanced downstream signaling. Understanding the complex phosphorylation patterns of IRS-1 is crucial to elucidating the factors contributing to insulin resistance and, ultimately, the pathogenesis of type 2 diabetes.
Insulin-like growth factor-1 (IGF-1) is a polypeptide growth factor with a variety of functions in both neuronal and non-neuronal cells. IGF-1 plays anti-apoptotic and other functions by activating multiple signaling pathways including Akt kinase, a serine/threonine kinase essential for cell survival. The nuclear transcription factor cAMP response element-binding protein (CREB) may also be involved although relationships between these two proteins in IGF-1 receptor signaling and protection is not clear, especially in neuronal cells.
IGF-1, in a concentration- and time-dependent manner, induces the activation/phosphorylation of Akt and CREB in PC12 cells by activating different signaling pathways. IGF-1 induced a sustained phosphorylation of Akt while only a transient one was seen for CREB. The phosphorylation of Akt is mediated by the PI3 kinase pathway while that of CREB is dependent on the activation of both MAPK kinase and p38 MAPK. Moreover, the stimulation of PKC attenuated the phosphorylation of Akt induced by IGF-1 while enhancing that of CREB. Survival assays with various kinase inhibitors suggested that the activation/phosphorylation of both Akt and CREB contributes to IGF-1 mediated cell survival in PC12 cells.
These data suggest that IGF-1 induced the activation of Akt and CREB using distinct pathways in PC12 cells.
Serine/threonine phosphorylation of IRS-1 might inhibit insulin signaling, but the relevant phosphorylation sites are difficult to identify in cultured cells and to validate in isolated tissues. Recently, we discovered that recombinant NH2-terminal Jun kinase phosphorylates IRS-1 at Ser307, which inhibits insulin-stimulated tyrosine phosphorylation of IRS-1. To monitor phosphorylation of Ser307 in various cell and tissue backgrounds, we prepared a phosphospecific polyclonal antibody designated αpSer307. This antibody revealed that TNF-α, IGF-1, or insulin stimulated phosphorylation of IRS-1 at Ser307 in 3T3-L1 preadipocytes and adipocytes. Insulin injected into mice or rats also stimulated phosphorylation of Ser307 on IRS-1 immunoprecipitated from muscle; moreover, Ser307 was phosphorylated in human muscle during the hyperinsulinemic euglycemic clamp. Experiments in 3T3-L1 preadipocytes and adipocytes revealed that insulin-stimulated phosphorylation of Ser307 was inhibited by LY294002 or wortmannin, whereas TNF-α–stimulated phosphorylation was inhibited by PD98059. Thus, distinct kinase pathways might converge at Ser307 to mediate feedback or heterologous inhibition of IRS-1 signaling to counterregulate the insulin response.
The authors previously reported that physiological light induces the tyrosine phosphorylation of insulin receptors (IRs), which leads to the activation of the phosphoinositide 3-kinase (PI3K) and Akt (serine/threonine protein kinase B) survival pathway in rod photoreceptor cells. Tissue-specific deletion of IRs from photoreceptors resulted in stress-induced photoreceptor degeneration. Insulin growth factor 1 receptor (IGF-1R) is highly related in sequence and structure to the IR and shares 70% sequence identity overall and 84% identity within the tyrosine kinase domain. The role of IGF-1R in photoreceptor function is unknown. In this study the authors examined IGF-1R signaling in rod outer segment (ROS) membranes.
IGF-1R localization was examined in the plasma and disc membranes of ROS. Activation of the IGF-1R/PI3K/Akt pathway was analyzed using specific antibodies against phospho-tyrosine, IGF-1R, and phospho-Akt. PI3K activity was determined in the anti-phospho-tyrosine and anti-IGF-1R immunoprecipitates. Glutathione-S-transferase fusion proteins containing two Src homology 2 (SH2) domains of the p85 subunit of PI3K and their mutants were used to study the molecular interaction between IGF-1R and p85. In vivo IGF-1R signaling was studied in rats exposed to physiological light or to constant light.
IGF-1R is predominately localized to plasma membranes of ROS. These studies indicate that light stress results in an increase in tyrosine phosphorylation of IGF-1R and an increase in PI3K enzyme activity in anti-phosphotyrosine and anti-IGF-1R immunoprecipitates of ROS and retinal homogenates. The authors observed that light stress induces tyrosine phosphorylation of IGF-1R in ROS membranes, which leads to the binding of p85 through N-SH2 and C-SH2 domains. Finally, the authors observed a significant activation of Akt in light-stressed retinas, indicating activation of the Akt survival pathway downstream of IGF-1R activation.
Light stress induced the activation of PI3K through activation and binding of IGF-1R, which leads to activation of the Akt survival pathway in photoreceptors.
A temporal framework linking circadian rhythms and clocks to aging rates identifies a specific window of target of rapamycin (TOR) signaling associated with growth hormone (GH) and insulin-like growth factor (IGF-1) (largely exclusive of insulin) in early sleep. IGF-1 signaling is released by growth hormone secretory peaks and downregulation of IGF-1 binding protein-1 resulting in activation of the mitogen activated protein kinase/extracellular signal response kinase (MAPK/ERK) and phosphoinositide 3-kinase-protein kinase B (PI3K-PKB/Akt) signaling pathways. Phosphorylation of Akt activates TOR which mediates the protein synthesis and growth functions of the GH axis. TOR activity is also associated with downregulated stress resistance, faster aging and reduced lifespan. IGF-1 signaling is terminated by falling GH and upregulation of IGF-1 binding proteins mediated by somatostatin and rising corticosteroids in later sleep. This suppresses PI3K-Akt signaling, thus activating the forkhead transcription factors (FOXOs) and stress-resistance pathways involved in promoting longevity. Thus, sleep appears to encompass both pathways currently identified as most relevant to aging and they toggle successively on the phosphorylation status of Akt. I propose a modified version of Pearl’s rate of living theory emphasizing the hard-wired antagonism of growth (TOR) and stress resistance (FOXO). The sleep association of TOR and FOXO in temporally separated windows and their sequential temporal deployment may change much of the way we think about aging and how to manipulate it.
Forkhead transcription factors; target of rapamycin; clocks; energy metabolism; rate of living; stress resistance; growth; sleep
In the present study, we investigated the potential role of insulin-like growth factor I (IGF-I) receptor (IGF-IR) in cell proliferation by overexpressing it in 32D myeloid progenitor cells. The overexpression of IGF-IR caused the transfectants to proliferate in response to IGF-I in the absence of insulin receptor substrate (IRS) expression. The activation of overexpressed wild-type IGF-IR, but not that of an ATP-binding mutant of IGF-IR, resulted in the increased tyrosine phosphorylation of several intracellular proteins, including SHC, Src homology 2-containing inositol-5-phosphatase, protein kinase C-δ, and Erk2. Grb2 association with SHC and mitogen-activated protein kinase (MAPK) activity was also enhanced in response to IGF-I stimulation. Interestingly, the stimulation of the IGF-IR transfectants with interleukin 4 (IL-4) also resulted in strong mitogenesis independent of IRS expression. Moreover, IGF-I and/or IL-4 induced long-term cell growth of the IGF-IR transfectants. IL-4 was able to synergize with IGF-I for DNA synthesis, even in the parental 32D cells and a pro-B-cell line, Baf3, indicating the physiological importance of the two growth factors in hematopoietic cell proliferation. IL-4 stimulation of the IGF-IR transfectants resulted in enhanced tyrosine phosphorylation of SHC, Erk2, and signal transducer and activator of transcription 6 (STAT6) proteins. Both IL-4 and IGF-I were able to induce c-myc early response gene expression, and this expression was maximal in the presence of both factors. Finally, we demonstrated that a MAPK kinase inhibitor was able to suppress mitogenesis of the IGF-IR transfectants in response to IGF-I and/or IL-4. Together, our results suggest that IL-4 synergizes with IGF-I for hematopoietic cell proliferation, likely through cross talk between SHC/Grb2/MAPK and STAT6 pathways and through c-myc gene up-regulation.
The tyrosine kinase activity of insulin receptor isolated from the skeletal muscle of NIDDM patients has previously been found to be decreased compared with the activity of receptor from nondiabetic subjects but the mechanism underlying this defect is unknown. Phosphorylation of receptor serine/threonine residues has been proposed to exert an inhibitory influence on receptor tyrosine kinase activity and Ser 1327 and Thr 1348 have been identified as specific sites of phosphorylation in the insulin receptor COOH terminal domain. To address the potential negative regulatory role of phosphorylation of these residues in vivo, we assessed the extent of phosphorylation of each site in insulin receptor isolated from the skeletal muscle of 12 NIDDM patients and 13 nondiabetic, control subjects. Phosphorylation of Ser 1327 and Thr 1348 was determined using antibodies that specifically recognize insulin receptor phosphorylated at these sites. In addition, a phosphotyrosine-specific antibody was used to monitor receptor tyrosine phosphorylation. The extent of insulin-induced tyrosine autophosphorylation was decreased in receptor isolated from diabetic versus nondiabetic muscle, thus confirming earlier reports. In contrast, there was no significant difference in the extent of phosphorylation of either Ser 1327 or Thr 1348 in receptor isolated from diabetic or nondiabetic muscle as assessed by immunoprecipitation (Ser 1327: 5.6 +/- 1.6% diabetics vs. 4.7 +/- 2.0% control; Thr 1348: 3.8 +/- 1.0% diabetics vs. 3.2 +/- 1.2% control). Moreover, within each group there was no correlation between the level of tyrosine kinase activity and the extent of serine/threonine phosphorylation. It is concluded that the stoichiometry of serine/threonine phosphorylation of insulin receptor in vivo is low, and that increased phosphorylation of Ser 1327 or Thr 1348 is not responsible for the decreased insulin receptor tyrosine kinase activity observed in the skeletal muscle of NIDDM patients.
Insulin-like growth factor I (IGF-I) is a mitogen for vascular smooth muscle cells (VSMC) and has been implicated in the development and progression of atherosclerosis. IGF binding proteins (IGFBPs) modify IGF-I actions independently of IGF binding, but a receptor-based mechanism by which they function has not been elucidated. We investigated the role of IGFBP-2 and receptor protein tyrosine phosphatase β (RPTPβ) in regulating IGF-I signaling and cellular proliferation. IGFBP-2 bound RPTPβ, which led to its dimerization and inactivation. This enhanced PTEN tyrosine phosphorylation and inhibited PTEN activity. Utilization of substrate trapping and phosphatase-dead mutants showed that RPTPβ bound specifically to PTEN and dephosphorylated it. IGFBP-2 knockdown led to decreased PTEN tyrosine phosphorylation and decreased AKT Ser473 activation. IGFBP-2 enhanced IGF-I-stimulated VSMC migration and proliferation. Analysis of aortas obtained from IGFBP-2−/− mice showed that RPTPβ was activated, and this was associated with inhibition of IGF-I stimulated AKT Ser473 phosphorylation and VSMC proliferation. These changes were rescued following administration of IGFBP-2. These findings present a novel mechanism for coordinate regulation of IGFBP-2 and IGF-I signaling functions that lead to stimulation of VSMC proliferation. The results have important implications for understanding how IGFBPs modulate the cellular response to IGF-I.
Insulin receptor substrate 1 (IRS-1) is the principle cellular substrate for insulin and insulin-like growth factor I (IGF-I) receptor signaling. After phosphorylation of tyrosine residues within the YMXM or YXXM motifs, IRS-1 associates with phosphatidylinositol-3 kinase (PI3K). This signaling pathway and the presence of an IRS-1-like molecule have been demonstrated in IRS-1-transfected and in nontransfected hematopoietic cell lines, respectively. IGF-I has been implicated in lymphocyte development and function, and recently, we showed that functional type-I IGF receptors are present on human thymocytes and peripheral T cells. In this study, we addressed IGF-I signal transduction in nontransformed, freshly isolated, human thymocytes, as well as in blood T cells. Using Western blot analysis, we found that IGF-I induced phosphorylation of a 160-180-kD protein (pp170) in human thymocytes and that phosphorylated pp170 becomes associated with PI3K and is recognized by anti-IRS-1. In blood T cells, this immunoreactive IRS-1 (irIRS-1) is less abundantly expressed than in thymocytes, as assessed with immunoblotting. As a consequence, phosphorylated pp170 was not or hardly detectable after stimulation with IGF-I, and irIRS-1 was not detected in PI3K immunoprecipitates from lysates of IGF-I-stimulated T cells. However, IGF-I induced the tyrosine phosphorylation of other cellular proteins, indicating that differential expression of irIRS-1 contributes to a distinct signaling pathway in T cells.
Insulin-like growth factor I (IGF-I) stimulates smooth muscle cell (SMC) proliferation, and the mitogen-activated protein kinase (MAPK) pathway plays an important role in mediating IGF-I–induced mitogenic signaling. Our prior studies have shown that recruitment of Src homology 2 domain tyrosine phosphatase (SHP-2) to the membrane scaffolding protein Src homology 2 domain–containing protein tyrosine phosphatase substrate-1 (SHPS-1) is required for IGF-I–dependent MAPK activation. The current studies were undertaken to define the upstream signaling components that are required for IGF-I–stimulated MAPK activation and the role of SHPS-1 in regulating this process. The results show that IGF-I–induced Shc phosphorylation and its subsequent binding to Grb2 is required for sustained phosphorylation of MAPK and increased cell proliferation in SMCs. Furthermore, for Shc to be phosphorylated in response to IGF-I requires that Shc must associate with SHPS-1 and this association is mediated in part by SHP-2. Preincubation of cells with a peptide that contains a phospho-tyrosine binding motif sequence derived from SHPS-1 inhibited IGF-I–stimulated SHP-2 transfer to SHPS-1, the association of Shc with SHPS-1, and IGF-I–dependent Shc phosphorylation. Expression of an SHPS-1 mutant that did not bind to Shc or SHP-2 resulted in decreased Shc and MAPK phosphorylation in response to IGF-I. In addition, SMCs expressing a mutant form of the β3 subunit of the αVβ3, which results in impairment of SHP-2 transfer to SHPS-1, also showed attenuated IGF-I–dependent Shc and MAPK phosphorylation. Further analysis showed that Shc and SHP-2 can be coimmunoprecipitated after IGF-I stimulation. A cell-permeable peptide that contained a polyproline sequence from Shc selectively inhibited Shc/SHP-2 association and impaired Shc but not SHP-2 binding to SHPS-1. Exposure to this peptide also inhibited IGF-I–stimulated Shc and MAPK phosphorylation. Cells expressing a mutant form of Shc with the four prolines substituted with alanines showed no Shc/SHPS-1 association in response to IGF-I. We conclude that SHPS-1 functions as an anchor protein that recruits both Shc and SHP-2 and that their recruitment is necessary for IGF-I–dependent Shc phosphorylation, which is required for an optimal mitogenic response in SMCs.
The eIF4E-binding proteins (4E-BPs) interact with translation initiation factor 4E to inhibit translation. Their binding to eIF4E is reversed by phosphorylation of several key Ser/Thr residues. In Drosophila, S6 kinase (dS6K) and a single 4E-BP (d4E-BP) are phosphorylated via the insulin and target of rapamycin (TOR) signaling pathways. Although S6K phosphorylation is independent of phosphoinositide 3-OH kinase (PI3K) and serine/threonine protein kinase Akt, that of 4E-BP is dependent on PI3K and Akt. This difference prompted us to examine the regulation of d4E-BP in greater detail. Analysis of d4E-BP phosphorylation using site-directed mutagenesis and isoelectric focusing-sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the regulatory interplay between Thr37 and Thr46 of d4E-BP is conserved in flies and that phosphorylation of Thr46 is the major phosphorylation event that regulates d4E-BP activity. We used RNA interference (RNAi) to target components of the PI3K, Akt, and TOR pathways. RNAi experiments directed at components of the insulin and TOR signaling cascades show that d4E-BP is phosphorylated in a PI3K- and Akt-dependent manner. Surprisingly, RNAi of dAkt also affected insulin-stimulated phosphorylation of dS6K, indicating that dAkt may also play a role in dS6K phosphorylation.
IGF-I stimulation of cell proliferation and c-Fos expression in skeletal muscle cells is markedly enhanced by dexamethasone. The effect of dexamethasone is not mediated by changes in IGF-binding proteins, as evidenced by similar effects of dexamethasone on the actions of insulin, PDGF-BB, and the IGF-I analogue long R3IGF-I. Dexamethasone also does not alter autocrine IGF-II secretion by muscle cells. To investigate the mechanism of the augmentation of IGF-I action, the effects of dexamethasone on intracellular IGF-I signaling pathways were determined. In dexamethasone-treated cells, the levels of IGF-I receptor tyrosine phosphorylation and receptor-associated phosphatidylinositol 3-kinase activity were increased. Dexamethasone-treated cells also showed increased and prolonged tyrosine phosphorylation of the Shc proteins. In contrast, dexamethasone decreased both tyrosine phosphorylation and expression of insulin receptor substrate 1 (IRS-1) and IRS-1-associated phosphatidylinositol 3-kinase activity. Thus, distinct signaling pathways activated by the IGF-I receptor in skeletal muscle cells are differentially regulated by dexamethasone. Potentiation of IGF-I action correlates with increased IGF-I receptor-associated phosphatidylinositol 3-kinase activity and tyrosine phosphorylation of Shc, but appears to be independent of activation of the IRS-1/phosphatidylinositol 3-kinase signaling pathway.
Elevated glucose concentrations have been reported to inhibit insulin receptor kinase activity. We studied the effects of high glucose on insulin action in Rat1 fibroblasts transfected with wild-type human insulin receptor (HIRcB) and a truncated receptor lacking the COOH-terminal 43 amino acids (delta CT). In both cell lines, 25 mM glucose impaired receptor and insulin receptor substrate-1 phosphorylation by 34%, but IGF-1 receptor phosphorylation was unaffected. Phosphatidylinositol 3-kinase activity and bromodeoxyuridine uptake were decreased by 85 and 35%, respectively. This was reversed by coincubation with a protein kinase C (PKC) inhibitor or microinjection of a PKC inhibitor peptide. Phosphopeptide mapping revealed that high glucose or PMA led to serine/threonine phosphorylation of similar peptides. Inhibition of the microtubule-associated protein (MAP) kinase cascade by the MAP kinase kinase inhibitor PD98059 did not reverse the impaired phosphorylation. We conclude that high glucose inhibits insulin action by inducing serine phosphorylation through a PKC-mediated mechanism at the level of the receptor at sites proximal to the COOH-terminal 43 amino acids. This effect is independent of activation of the MAP kinase cascade. Proportionately, the impairment of insulin receptor substrate-1 tyrosine phosphorylation is greater than that of the insulin receptor resulting in attenuated phosphatidylinositol 3-kinase activation and mitogenic signaling.
The serine/threonine kinase PKB/Akt plays essential role in various cellular processes including cell growth and proliferation, metabolism and cell survival. The importance of the Akt pathway is highlighted by the mutation of various components of the pathway such as the PTEN and PI3-kinase (P110α) in human cancers. In this paper, we employed an RNA interference library targeting all human kinases to screen for kinases involved in the regulation of Akt activation, in particular serine 473 phosphorylation. Here, we transfected the MDA-MB 468 breast cell line with the human kinome siRNA library and measured Akt activation using an antibody specific for phosphoserine 473 of Akt.
The screen revealed that phosphorylation of Akt(ser473) can be regulated by more than 90 kinases. Interestingly, phosphorylation of Akt(ser473), but not thr308, can be severely reduced by inhibition of Choline kinase activity via siRNA or small molecule inhibitors. We show here that the regulation of Akt phosphorylation by Choline kinase is PI3K-independent. In addition, xenograft tumors treated with Choline kinase inhibitors demonstrated a statistically significant decrease in Akt(ser473) phosphorylation. Importantly, the reduction in phosphorylation correlates with regression of these xenograft tumors in the mouse model.
High Choline kinase expression and activity has previously been implicated in tumor development and metastasis. The mechanism by which Choline kinase is involved in tumor formation is still not fully resolved. From our data, we proposed that Choline kinase plays a key role in regulating Akt(ser473) phosphorylation, thereby promoting cell survival and proliferation.
The insulin-like growth factor 1 receptor (IGF-1R) plays numerous crucial roles in cancer biology. The majority of knowledge on IGF-1R signaling is concerned with its role in the activation of the canonical phosphatidyl inositol-3 kinase (PI3K)/Akt and MAPK/ERK pathways. However, the role of IGF-1R ubiquitination in modulating IGF-1R function is an area of current research. In light of this we sought to determine the relationship between IGF-1R phosphorylation, ubiquitination, and modulation of growth signals.
Wild type and mutant constructs of IGF-1R were transfected into IGF-1R null fibroblasts. IGF-1R autophosphorylation and ubiquitination were determined by immunoprecipitation and western blotting. IGF-1R degradation and stability was determined by cyclohexamide-chase assay in combination with lysosome and proteasome inhibitors.
IGF-1R autophosphorylation was found to be an absolute requirement for receptor ubiquitination. Deletion of C-terminal domain had minimal effect on IGF-1 induced receptor autophosphorylation, however, ubiquitination and ERK activation were completely abolished. Cells expressing kinase impaired IGF-1R, exhibited both receptor ubiquitination and ERK phosphorylation, however failed to activate Akt. While IGF-1R mutants with impaired PI3K/Akt signaling were degraded mainly by the proteasomes, the C-terminal truncated one was exclusively degraded through the lysosomal pathway.
Our data suggest important roles of ubiquitination in mediating IGF-1R signaling and degradation. Ubiquitination of IGF-1R requires receptor tyrosine kinase activity, but is not involved in Akt activation. In addition we show that the C-terminal domain of IGF-1R is a necessary requisite for ubiquitination and ERK phosphorylation as well as for proteasomal degradation of the receptor.
Protein phosphorylation occurs in certain sequence/structural contexts that are still incompletely understood. The amino acids surrounding the phosphorylated residues are important in determining the binding of the kinase to the protein sequence. Upon phosphorylation these sequences also determine the binding of certain domains that specifically bind to phosphorylated sequences. Thus far, such ‘motifs’ have been identified through alignment of a limited number of well identified kinase substrates.
Experimentally determined phosphorylation sites from Human Protein Reference Database were used to identify 1,167 novel serine/threonine or tyrosine phosphorylation motifs using a computational approach. We were able to statistically validate a number of these novel motifs based on their enrichment in known phosphopeptides datasets over phosphoserine/threonine/tyrosine peptides in the human proteome. There were 299 novel serine/threonine or tyrosine phosphorylation motifs that were found to be statistically significant. Several of the novel motifs that we identified computationally have subsequently appeared in large datasets of experimentally determined phosphorylation sites since we initiated our analysis. Using a peptide microarray platform, we have experimentally evaluated the ability of casein kinase I to phosphorylate a subset of the novel motifs discovered in this study. Our results demonstrate that it is feasible to identify novel phosphorylation motifs through large phosphorylation datasets. Our study also establishes peptide microarrays as a novel platform for high throughput kinase assays and for the validation of consensus motifs. Finally, this extended catalog of phosphorylation motifs should assist in a systematic study of phosphorylation networks in signal transduction pathways.
Phosphorylation; Motifs; Peptide array
Skeletal muscle expresses high levels of integrin-linked kinase (ILK), predominantly at myotendinous junctions (MTJs) and costameres. ILK binds the cytoplasmic domain of β1 integrin and mediates phosphorylation of protein kinase B (PKB)/Akt, which in turn plays a central role during skeletal muscle regeneration. We show that mice with a skeletal muscle–restricted deletion of ILK develop a mild progressive muscular dystrophy mainly restricted to the MTJs with detachment of basement membranes and accumulation of extracellular matrix. Endurance exercise training enhances the defects at MTJs, leads to disturbed subsarcolemmal myofiber architecture, and abrogates phosphorylation of Ser473 as well as phosphorylation of Thr308 of PKB/Akt. The reduction in PKB/Akt activation is accompanied by an impaired insulin-like growth factor 1 receptor (IGF-1R) activation. Coimmunoprecipitation experiments reveal that the β1 integrin subunit is associated with the IGF-1R in muscle cells. Our data identify the β1 integrin–ILK complex as an important component of IGF-1R/insulin receptor substrate signaling to PKB/Akt during mechanical stress in skeletal muscle.
Activation of protein kinase B (PKB) by growth factors and hormones has been demonstrated to proceed via phosphatidylinositol 3-kinase (PI3-kinase). In this report, we show that PKB can also be activated by PKA (cyclic AMP [cAMP]-dependent protein kinase) through a PI3-kinase-independent pathway. Although this activation required phosphorylation of PKB, PKB is not likely to be a physiological substrate of PKA since a mutation in the sole PKA consensus phosphorylation site of PKB did not abolish PKA-induced activation of PKB. In addition, mechanistically, this activation was different from that of growth factors since it did not require phosphorylation of the S473 residue, which is essential for full PKB activation induced by insulin. These data were supported by the fact that mutation of residue S473 of PKB to alanine did not prevent it from being activated by forskolin. Moreover, phosphopeptide maps of overexpressed PKB from COS cells showed differences between insulin- and forskolin-stimulated cells that pointed to distinct activation mechanisms of PKB depending on whether insulin or cAMP was used. We looked at events downstream of PKB and found that PKA activation of PKB led to the phosphorylation and inhibition of glycogen synthase kinase-3 (GSK-3) activity, a known in vivo substrate of PKB. Overexpression of a dominant negative PKB led to the loss of inhibition of GSK-3 in both insulin- and forskolin-treated cells, demonstrating that PKB was responsible for this inhibition in both cases. Finally, we show by confocal microscopy that forskolin, similar to insulin, was able to induce translocation of PKB to the plasma membrane. This process was inhibited by high concentrations of wortmannin (300 nM), suggesting that forskolin-induced PKB movement may require phospholipids, which are probably not generated by class I or class III PI3-kinase. However, high concentrations of wortmannin did not abolish PKB activation, which demonstrates that translocation per se is not important for PKA-induced PKB activation.
The receptor for insulin-like growth factor I (IGF-IR) controls normal and pathological growth of cells. DNA repair pathways represent an unexplored target through which the IGF-IR signaling system might support pathological growth leading to cellular transformation. However, this study demonstrates that IGF-I stimulation supports homologous recombination-directed DNA repair (HRR). This effect involves an interaction between Rad51 and the major IGF-IR signaling molecule, insulin receptor substrate 1 (IRS-1). The binding occurs within the cytoplasm, engages the N-terminal domain of IRS-1, and is attenuated by IGF-I-mediated IRS-1 tyrosine phosphorylation. In the absence of IGF-I stimulation, or if mutated IGF-IR fails to phosphorylate IRS-1, localization of Rad51 to the sites of damaged DNA is diminished. These results point to a direct role of IRS-1 in HRR and suggest a novel role for the IGF-IR/IRS-1 axis in supporting the stability of the genome.
AGC kinases, including the three Akt (protein kinase B) isoforms, protein kinase A (PKA) and all protein kinase C (PKC) isoforms, require activation loop phosphorylation (threonine 308 in Akt1) as well as phosphorylation of a C-terminal residue (serine 473 in Akt1) for catalytic activity and phosphorylation of downstream targets. Conversely, phosphatases reverse these phosphorylations. Virtually all cellular processes are affected by AGC kinases, a circumstance that has led to intense scrutiny of the molecular mechanisms that regulate phosphorylation of these kinases. Here, we review a new layer of control of phosphorylation in Akt, PKA and PKC pointing to ATP binding pocket occupancy as a means to decelerate dephosphorylation of these and, potentially, other kinases. This additional level of kinase regulation opens the door to search for new functional motifs for the rational design of non-ATP-competitive kinase inhibitors that discriminate within and between protein kinase families.
inhibitors hijacking kinase activation; activation loop phosphorylation; dephosphorylation; phosphatase resistance; PKA; PKB; PKC
The C. elegans insulin/IGF-1 signaling (IIS) cascade plays a central role in the regulation of lifespan, dauer diapause, metabolism and stress response. The major regulatory control of IIS is through phosphorylation of its components by serine/threonine-specific protein kinases. In a RNAi screen for serine/threonine protein phosphatases that counter-balance the effect of the kinases in the IIS pathway, we identified pptr-1, a B56 regulatory subunit of the PP2A holoenzyme. Modulation of pptr-1 affects phenotypes associated with the IIS pathway including lifespan, dauer, stress resistance and fat storage. We show that PPTR-1 functions by regulating worm AKT-1 phosphorylation at Thr 350. With striking conservation, mammalian B56β regulates Akt phosphorylation at Thr 308 in 3T3-L1 adipocytes. In C. elegans, this modulation ultimately leads to changes in subcellular localization and transcriptional activity of the forkhead transcription factor DAF-16. This study reveals a conserved role for the B56 regulatory subunit in modulating insulin signaling through AKT dephosphorylation and thereby has widespread implications in cancer and diabetes research.
The protein kinase B (PKB)/Akt family of serine kinases is rapidly activated following agonist-induced stimulation of phosphoinositide 3-kinase (PI3K). To probe the molecular events important for the activation process, we employed two distinct models of posttranslational inducible activation and membrane recruitment. PKB induction requires phosphorylation of two critical residues, threonine 308 in the activation loop and serine 473 near the carboxyl terminus. Membrane localization of PKB was found to be a primary determinant of serine 473 phosphorylation. PI3K activity was equally important for promoting phosphorylation of serine 473, but this was separable from membrane localization. PDK1 phosphorylation of threonine 308 was primarily dependent upon prior serine 473 phosphorylation and, to a lesser extent, localization to the plasma membrane. Mutation of serine 473 to alanine or aspartic acid modulated the degree of threonine 308 phosphorylation in both models, while a point mutation in the substrate-binding region of PDK1 (L155E) rendered PDK1 incapable of phosphorylating PKB. Together, these results suggest a mechanism in which 3′ phosphoinositide lipid-dependent translocation of PKB to the plasma membrane promotes serine 473 phosphorylation, which is, in turn, necessary for PDK1-mediated phosphorylation of threonine 308 and, consequentially, full PKB activation.
A wide variety of biological activities including the major metabolic actions of insulin is regulated by phosphatidylinositol (PI) 3-kinase. However, the downstream effectors of the various signaling pathways that emanate from PI 3-kinase remain unclear. Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, is thought to be one such downstream effector. A mutant Akt (Akt-AA) in which the phosphorylation sites (Thr308 and Ser473) targeted by growth factors are replaced by alanine has now been shown to lack protein kinase activity and, when overexpressed in CHO cells or 3T3-L1 adipocytes with the use of an adenovirus vector, to inhibit insulin-induced activation of endogenous Akt. Akt-AA thus acts in a dominant negative manner in intact cells. Insulin-stimulated protein synthesis, which is sensitive to wortmannin, a pharmacological inhibitor of PI 3-kinase, was abolished by overexpression of Akt-AA without an effect on amino acid transport into the cells, suggesting that Akt is required for insulin-stimulated protein synthesis. Insulin activation of p70 S6 kinase was inhibited by ∼75% in CHO cells and ∼30% in 3T3-L1 adipocytes, whereas insulin-induced activation of endogenous Akt was inhibited by 80 to 95%, by expression of Akt-AA. Thus, Akt activity appears to be required, at least in part, for insulin stimulation of p70 S6 kinase. However, insulin-stimulated glucose uptake in both CHO cells and 3T3-L1 adipocytes was not affected by overexpression of Akt-AA, suggesting that Akt is not required for this effect of insulin. These data indicate that Akt acts as a downstream effector in some, but not all, of the signaling pathways downstream of PI 3-kinase.
IRS-1 (insulin receptor substrate 1) is a principal insulin receptor substrate that undergoes tyrosine phosphorylation during insulin stimulation. It contains over 20 potential tyrosine phosphorylation sites, and we suspect that multiple insulin signals are enabled when the activated insulin receptor kinase phosphorylates several of them. Tyrosine-phosphorylated IRS-1 binds specifically to various cellular proteins containing Src homology 2 (SH2) domains (SH2 proteins). We identified some of the tyrosine residues of IRS-1 that undergo insulin-stimulated phosphorylation by the purified insulin receptor and in intact cells during insulin stimulation. Automated sequencing and manual radiosequencing revealed the phosphorylation of tyrosine residues 460, 608, 628, 895, 939, 987, 1172, and 1222; additional sites remain to be identified. Immobilized SH2 domains from the 85-kDa regulatory subunit (p85 alpha) of the phosphatidylinositol 3'-kinase bind preferentially to tryptic phosphopeptides containing Tyr(P)-608 and Tyr(P)-939. By contrast, the SH2 domain in GRB2 and the amino-terminal SH2 domain in SHPTP2 (Syp) specifically bind to Tyr(P)-895 and Tyr(P)-1172, respectively. These results confirm the p85 alpha recognizes YMXM motifs and suggest that GRB2 prefers a phosphorylated YVNI motif, whereas SHPTP2 (Syp) binds to a phosphorylated YIDL motif. These results extend the notion that IRS-1 is a multisite docking protein that engages various downstream regulatory elements during insulin signal transmission.