Phorbol ester treatment increases the exocytic rate of the transferrin receptor recycling pathway independent of serine-24 phosphorylation

In Chinese hamster ovary (CHO) fibroblast cells the protein kinase C activating phorbol ester, phorbol myristate acetate (PMA), stimulates an increase in cell surface transferrin receptor (TR) expression by increasing the exocytic rate of the recycling pathway. The human TR expressed in CHO cells is similarly affected by PMA treatment. A mutant human TR in which the major protein kinase C phosphorylation site, serine 24, has been replaced with the non-phosphorylatable amino acid glycine has been constructed to investigate the role of receptor phosphorylation in the PMA induced up-regulation. The Gly-24- substituted receptor binds, internalizes, and recycles Tf. Furthermore, the altered receptor mediates cellular Fe accumulation from diferric- Tf, thereby fulfilling the receptor's major biological role. The Gly-24 TR behaves identically to the wild-type TR when cells are treated with PMA. Therefore, Ser-24 phosphorylation is not required for the PMA- induced redistribution of the human TR expressed in CHO cells. The increased TR expression on the cell surface after PMA treatment results from an increase in the rate of exocytosis of the recycling receptors. No change in the endocytic rate or the size of the recycling receptor pool was observed. These results indicate that the PMA effect on the TR surface expression may result from a more general perturbation of membrane trafficking rather than a specific modulation of the TR.     

Phosphorylation of the yeast choline kinase by protein kinase C. Identification of Ser25 and Ser30 as major sites of phosphorylation

The Saccharomyces cerevisiae CKI1-encoded choline kinase catalyzes the committed step in phosphatidylcholine synthesis via the Kennedy pathway. The enzyme is phosphorylated on multiple serine residues, and some of this phosphorylation is mediated by protein kinase A. In this work we examined the hypothesis that choline kinase is also phosphorylated by protein kinase C. Using choline kinase as a substrate, protein kinase C activity was dose- and time-dependent and dependent on the concentrations of choline kinase (K(m) = 27 microg/ml) and ATP (K(m) = 15 microM). This phosphorylation, which occurred on a serine residue, was accompanied by a 1.6-fold stimulation of choline kinase activity. The synthetic peptide SRSSSQRRHS (V5max/K(m) = 17.5 mm(-1) micromol min(-1) mg(-1)) that contains the protein kinase C motif for Ser25 was a substrate for protein kinase C. A Ser25 to Ala (S25A) mutation in choline kinase resulted in a 60% decrease in protein kinase C phosphorylation of the enzyme. Phosphopeptide mapping analysis of the S25A mutant enzyme confirmed that Ser25 was a protein kinase C target site. In vivo the S25A mutation correlated with a decrease (55%) in phosphatidylcholine synthesis via the Kennedy pathway, whereas an S25D phosphorylation site mimic correlated with an increase (44%) in phosphatidylcholine synthesis. Although the S25A (protein kinase C site) mutation did not affect the phosphorylation of choline kinase by protein kinase A, the S30A (protein kinase A site) mutation caused a 46% reduction in enzyme phosphorylation by protein kinase C. A choline kinase synthetic peptide (SQRRHSLTRQ) containing Ser30 was a substrate (V(max)/K(m) = 3.0 mm(-1) micromol min(-1) mg(-1)) for protein kinase C. Comparison of phosphopeptide maps of the wild type and S30A mutant choline kinase enzymes phosphorylated by protein kinase C confirmed that Ser30 was also a target site for protein kinase C.     

Phosphorylation of human keratin 18 serine 33 regulates binding to 14-3-3 proteins.

Members of the 14-3-3 protein family bind the human intermediate filament protein keratin 18 (K18) in vivo, in a cell-cycle- and phosphorylation-dependent manner. We identified K18 Ser33 as an interphase phosphorylation site, which increases its phosphorylation during mitosis in cultured cells and regenerating liver, and as an in vitro cdc2 kinase phosphorylation site. Comparison of wild-type versus K18 Ser33-->Ala/Asp transfected cells showed that K18 Ser33 phosphorylation is essential for the association of K18 with 14-3-3 proteins, and plays a role in keratin organization and distribution. Mutation of another K18 major phosphorylation site (Ser52) or K18 glycosylation sites had no effect on the binding of K18 to 14-3-3 proteins. The K18 phospho-Ser33 motif is different from several 14-3-3-binding phosphomotifs already described. Antibodies that are specific to K18 phospho-Ser33 or phospho-Ser52 show that although Ser52 and Ser33 phosphorylated K18 molecules manifest partial colocalization, these phosphorylation events reside predominantly on distinct K18 molecules. Our results demonstrate a unique K18 phosphorylation site that is necessary but not sufficient for K18 binding to 14-3-3 proteins. This binding is likely to involve one or more mitotic events coupled to K18 Ser33 phosphorylation, and plays a role in keratin subcellular distribution. Physiological Ser52 or Ser33 phosphorylation on distinct K18 molecules suggests functional compartmentalization of these modifications.     

Oxidative stress employs phosphatidyl inositol 3-kinase and ERK signalling pathways to activate cAMP phosphodiesterase-4D3 (PDE4D3) through multi-site phosphorylation at Ser239 and Ser579

RAW macrophages, which express the PDE4D3 and PDE4D5 cAMP phosphodiesterase isoforms, exhibited increased PDE4 activity when challenged with H2O2 in a fashion that was negated by treatment with the cell permeant antioxidant, N-acetyl cysteine and by diphenyleneiodonium chloride, an inhibitor of NADPH oxidase. In Cos1 cells transfected to express PDE4D3, challenge with H2O2 caused a rapid increase in both the activity and phosphorylation of PDE4D3. Lysates from H2O2-treated COS cells caused the phosphorylation of purified, recombinant PDE4D3 at two sites. One was the established ERK phosphorylation site at Ser579, located at the extreme C-terminus of the catalytic unit, and the other was a novel site at Ser239, located at the extreme N-terminus of the catalytic unit. Double Ser239Ala:Ser579Ala mutation of PDE4D3 prevented its H2O2-dependent phosphorylation both in vitro and in intact COS cells. Phosphorylation of PDE4D3 at Ser579 was ablated by treating COS cells with the MEK inhibitor, PD98059, which also negated activation. The activity of the Ser239Ala:Ser579Ala double mutant, and the Ser579Ala single PDE4D3 mutant was unaffected by H2O2 challenge of COS cells, whilst the Ser239Ala mutant was inhibited. Wortmannin inhibited the H2O2-dependent phosphorylation of PDE4D3 in COS cells by around 50%, whilst it fully ablated phosphorylation at Ser239 as well as ablating activation of PDE4D3. Neither immunodepletion of p70S6 kinase nor siRNA-mediated knockdown of mTor inhibited the H2O2-dependent phosphorylation of PDE4D3 at Ser239. Activation of PDE4D3 by challenge with H2O2 was not additive with activation through protein kinase A (PKA)-mediated phosphorylation of PDE4D3. Challenge with H2O2 did not alter PKA-mediated phosphorylation of PDE4D3 at Ser54. H2O2 dependent phosphorylation of PDE4D3, at Ser239 and Ser579, did not alter the sensitivity of PDE4D3 to inhibition by the selective PDE4 inhibitor, rolipram. An unknown protein kinase acting downstream of phosphatidyl inositol 3-kinase phosphorylates PDE4D3 at Ser239. This switches the effect of phosphorylation by ERK at Ser579 from inhibition to activation. We propose that phosphorylation at Ser239 attenuates interaction between either UCR2 or the UCR1/UCR2 module and the PDE4 catalytic unit so as to re-programme the functional outcome effect of phosphorylation by ERK. We identify a novel process through which reactive oxygen species activate long PDE4 isoforms so as to reduce cAMP levels and thereby promote inflammatory responses.     

Mutants at Ser277 of Xenopus cdc2 protein kinase induce oocyte maturation in the absence of the positive regulatory phosphorylation site Thr161.

The cdc2 protein kinase is an important regulatory protein for both meiosis and mitosis. Previously, we demonstrated that simultaneous mutation of Thr14-->Ala14 and Tyr15-->Phe15 in the Xenopus cdc2 protein results in an activated cdc2 mutant that induces maturation in resting oocytes. In addition, we confirmed the importance of the positive regulatory phosphorylation site, Thr161, by demonstrating that cdc2 mutants containing additional mutations of Thr161-->Ala161 or Glu161 are inactive in the induction of oocyte maturation. Here, we have analyzed the importance of an additional putative cdc2 phosphorylation site,Ser277. Single mutation of Ser277-->Asp277 or Ala277 had no effect on activity, and these mutants were unable to induce Xenopus oocyte maturation. However, the double mutant Ala161/Asp277 was capable of inducing oocyte maturation, suggesting that mutation of Ser277-->Asp277 could compensate for the mutation of Thr161-->Ala161. The Asp277 mutation could also compensate for the Ala161 mutation in the background of the activating mutations Ala14/Phe15. Although mutants containing the compensatory Ala161 and Asp277 mutations were capable of inducing oocyte maturation, these mutant cdc2 proteins lacked detectable in vitro kinase activity. Tryptic phosphopeptide mapping of mutant cdc2 protein and comparison with in vitro synthesized peptides indicated that Ser277 is not a major site of phosphorylation in Xenopus oocytes; however, we cannot rule out the possibility of phosphorylation at this site in a biologically active subpopulation of cdc2 molecules. The data presented here, together with prior reports of Ser277 phosphorylation in somatic cells, suggest an important role for Ser277 in the regulation of cdc2 activity. The regulatory role of Ser277 most likely involves its indirect effects on the nearby residue Arg275, which participates in a structurally important ion pair with Glu173, which lies in the same loop as Thr161 in the cdc2 protein.     

Regulation of multisite phosphorylation and 14-3-3 binding of AS160 in response to IGF-1, EGF, PMA and AICAR

AS160 (Akt substrate of 160 kDa) mediates insulin-stimulated GLUT4 (glucose transporter 4) translocation, but is widely expressed in insulin-insensitive tissues lacking GLUT4. Having isolated AS160 by 14-3-3-affinity chromatography, we found that binding of AS160 to 14-3-3 isoforms in HEK (human embryonic kidney)-293 cells was induced by IGF-1 (insulin-like growth factor-1), EGF (epidermal growth factor), PMA and, to a lesser extent, AICAR (5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside). AS160-14-3-3 interactions were stabilized by chemical cross-linking and abolished by dephosphorylation. Eight residues on AS160 (Ser318, Ser341, Thr568, Ser570, Ser588, Thr642, Ser666 and Ser751) were differentially phosphorylated in response to IGF-1, EGF, PMA and AICAR. The binding of 14-3-3 proteins to HA-AS160 (where HA is haemagglutinin) was markedly decreased by mutation of Thr642 and abolished in a Thr642Ala/Ser341Ala double mutant. The AGC (protein kinase A/protein kinase G/protein kinase C-family) kinases RSK1 (p90 ribosomal S6 kinase 1), SGK1 (serum- and glucocorticoid-induced protein kinase 1) and PKB (protein kinase B) displayed distinct signatures of AS160 phosphorylation in vitro: all three kinases phosphorylated Ser318, Ser588 and Thr642; RSK1 also phosphorylated Ser341, Ser751 and to a lesser extent Thr568; and SGK1 phosphorylated Thr568 and Ser751. AMPK (AMP-activated protein kinase) preferentially phosphorylated Ser588, with less phosphorylation of other sites. In cells, the IGF-1-stimulated phosphorylations, and certain EGF-stimulated phosphorylations, were inhibited by PI3K (phosphoinositide 3-kinase) inhibitors, whereas the RSK inhibitor BI-D1870 inhibited the PMA-induced phosphorylations. The expression of LKB1 in HeLa cells and the use of AICAR in HEK-293 cells promoted phosphorylation of Ser588, but only weak Ser341 and Thr642 phosphorylations and binding to 14-3-3s. Paradoxically however, phenformin activated AMPK without promoting AS160 phosphorylation. The IGF-1-induced phosphorylation of the novel phosphorylated Ser666-Pro site was suppressed by AICAR, and by combined mutation of a TOS (mTOR signalling)-like sequence (FEMDI) and rapamycin. Thus, although AS160 is a common target of insulin, IGF-1, EGF, PMA and AICAR, these stimuli induce distinctive patterns of phosphorylation and 14-3-3 binding, mediated by at least four protein kinases.     

The role of DOC-2/DAB2 protein phosphorylation in the inhibition of AP-1 activity. An underlying mechanism of its tumor-suppressive function in prostate cancer.

DOC-2/DAB2, a novel phosphoprotein with signal-transducing capability, inhibits human prostatic cancer cells (Tseng, C.-P., Ely, B. D., Li, Y., Pong, R.-C., and Hsieh, J.-T. (1998) Endocrinology 139, 3542-3553). However, its mechanism of action is not understood completely. This study delineates the functional significance of DOC-2/DAB2 protein phosphorylation and demonstrates that in vivo activation of protein kinase C (PKC) by 12-O-tetradecanoylphorbol-13-acetate (TPA) induces DOC-2/DAB2 phosphorylation, including a serine residue at position 24. Mutation of Ser(24) to Ala reduced DOC-2/DAB2 phosphorylation by PKC. Using a synthetic Ser(24) peptide (APS(24)KKEKKKGSEKTD) or recombinant DOC-2/DAB2 as substrates, PKCbetaII, PKCgamma, and PKCdelta (but not casein kinase II) directly phosphorylated Ser(24) in vitro. This indicates that DOC-2/DAB2 is a PKC-specific substrate. Since expression of wild-type DOC-2/DAB2, but not the S24A mutant, inhibited TPA-induced AP-1 activity in prostatic epithelial cells, phosphorylation of Ser(24) appears to play a critical role in modulating TPA-induced AP-1 activity. Taken together, these data suggest that PKC-regulated phosphorylation of DOC-2/DAB2 protein may help its growth inhibitory function.     

Phosphorylation of Saccharomyces cerevisiae CTP synthetase at Ser424 by protein kinases A and C regulates phosphatidylcholine synthesis by the CDP-choline pathway

The Saccharomyces cerevisiae URA7-encoded CTP synthetase is phosphorylated and stimulated by protein kinases A and C. Previous studies have revealed that Ser424 is the target site for protein kinase A. Using a purified S424A mutant CTP synthetase enzyme, we examined the effect of Ser424 phosphorylation on protein kinase C phosphorylation. The S424A mutation in CTP synthetase caused a 50% decrease in the phosphorylation of the enzyme by protein kinase C and an 80% decrease in the stimulatory effect on CTP synthetase activity by protein kinase C. The S424A mutation caused increases in the apparent Km values of CTP synthetase and ATP of 20-and 2-fold, respectively, in the protein kinase C reaction. The effect of the S424A mutation on the phosphorylation reaction was dependent on time and protein kinase C concentration. A CTP synthetase synthetic peptide (SLGRKDSHSA) containing Ser424 was a substrate for protein kinase C. Comparison of phosphopeptide maps of the wild type and S424A mutant CTP synthetase enzymes phosphorylated by protein kinases A and C indicated that Ser424 was also a target site for protein kinase C. Phosphorylation of Ser424 accounted for 10% of the total phosphorylation of CTP synthetase by protein kinase C. The incorporation of [methyl-3H]choline into phosphocholine, CDP-choline, and phosphatidylcholine in cells carrying the S424A mutant CTP synthetase enzyme was reduced by 48, 32, and 46%, respectively, when compared with control cells. These data indicated that phosphorylation of Ser424 by protein kinase A or by protein kinase C was required for maximum phosphorylation and stimulation of CTP synthetase and that the phosphorylation of this site played a role in the regulation of phosphatidylcholine synthesis by the CDP-choline pathway.     

Molecular mechanism of the inhibition of phospholipase C beta 3 by protein kinase C

Activation of protein kinase C (PKC) can result from stimulation of the receptor-G protein-phospholipase C (PLCbeta) pathway. In turn, phosphorylation of PLCbeta by PKC may play a role in the regulation of receptor-mediated phosphatidylinositide (PI) turnover and intracellular Ca(2+) release. Activation of endogenous PKC by phorbol 12-myristate 13-acetate inhibited both Galpha(q)-coupled (oxytocin and M1 muscarinic) and Galpha(i)-coupled (formyl-Met-Leu-Phe) receptor-stimulated PI turnover by 50-100% in PHM1, HeLa, COSM6, and RBL-2H3 cells expressing PLCbeta(3). Activation of conventional PKCs with thymeleatoxin similarly inhibited oxytocin or formyl-Met-Leu-Phe receptor-stimulated PI turnover. The PKC inhibitory effect was also observed when PLCbeta(3) was stimulated directly by Galpha(q) or Gbetagamma in overexpression assays. PKC phosphorylated PLCbeta(3) at the same predominant site in vivo and in vitro. Peptide sequencing of in vitro phosphorylated recombinant PLCbeta(3) and site-directed mutagenesis identified Ser(1105) as the predominant phosphorylation site. Ser(1105) is also phosphorylated by protein kinase A (PKA; Yue, C., Dodge, K. L., Weber, G., and Sanborn, B. M. (1998) J. Biol. Chem. 273, 18023-18027). Similar to PKA, the inhibition by PKC of Galpha(q)-stimulated PLCbeta(3) activity was completely abolished by mutation of Ser(1105) to Ala. In contrast, mutation of Ser(1105) or Ser(26), another putative phosphorylation target, to Ala had no effect on inhibition of Gbetagamma-stimulated PLCbeta(3) activity by PKC or PKA. These data indicate that PKC and PKA act similarly in that they inhibit Galpha(q)-stimulated PLCbeta(3) as a result of phosphorylation of Ser(1105). Moreover, PKC and PKA both inhibit Gbetagamma-stimulated activity by mechanisms that do not involve Ser(1105).     

Identification of Ser424 as the protein kinase A phosphorylation site in CTP synthetase from Saccharomyces cerevisiae.

The URA7-encoded CTP synthetase [EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)] in the yeast Saccharomyces cerevisiae is phosphorylated on a serine residue and stimulated by cAMP-dependent protein kinase (protein kinase A) in vitro. In vivo, the phosphorylation of CTP synthetase is mediated by the RAS/cAMP pathway. In this work, we examined the hypothesis that amino acid residue Ser424 contained in a protein kinase A sequence motif in the URA7-encoded CTP synthetase is the target site for protein kinase A. A CTP synthetase synthetic peptide (SLGRKDSHSA) containing the protein kinase A motif was a substrate (Km = 30 microM) for protein kinase A. This peptide also inhibited (IC50 = 45 microM) the phosphorylation of purified wild-type CTP synthetase by protein kinase A. CTP synthetase with a Ser424 --> Ala (S424A) mutation was constructed by site-directed mutagenesis. The mutated enzyme was not phosphorylated in response to the activation of protein kinase A activity in vivo. Purified S424A mutant CTP synthetase was not phosphorylated and stimulated by protein kinase A. The S424A mutant CTP synthetase had reduced Vmax and elevated Km values for ATP and UTP when compared with the protein kinase A-phosphorylated wild-type enzyme. The specificity constants for ATP and UTP for the S424A mutant CTP synthetase were 4.2- and 2.9-fold lower, respectively, when compared with that of the phosphorylated enzyme. In addition, the S424A mutant enzyme was 2.7-fold more sensitive to CTP product inhibition when compared with the phosphorylated wild-type enzyme. These data indicated that the protein kinase A target site in CTP synthetase was Ser424 and that the phosphorylation of this site played a role in the regulation of CTP synthetase activity.     

Phosphorylation of serine 526 is required for MEKK3 activity, and association with 14-3-3 blocks dephosphorylation

MAPK/ERK kinase kinase 3 (MEKK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that functions upstream of the MAP kinases and IkappaB kinase. Phosphorylation is believed to be a critical component for MEKK3-dependent signal transduction, but little is known about the phosphorylation sites of this MAP3K. To address this question, point mutations were introduced in the activation loop (T-loop), substituting alanine for serine or threonine, and the mutants were transfected into HEK293 Epstein-Barr virus nuclear antigen cells. MEKK3-dependent activation of an NF-kappaB reporter gene as well as ERK, JNK, and p38 MAP kinases correlated with a requirement for serine at position 526. Constitutively active mutants of MEKK3, consisting of S526D and S526E, were capable of activating a NF-kappaB luciferase reporter gene as well as ERK and MEK, suggesting that a negative charge at Ser526 was necessary for MEKK3 activity and implicating Ser526 as a phosphorylation site. An antibody was developed that specifically recognized phospho-Ser526 of MEKK3 but did not recognize the S526A point mutant. The catalytically inactive (K391M) mutant of MEKK3 was not phosphorylated at Ser526, indicating that phosphorylation of Ser526 occurs via autophosphorylation. Endogenous MEKK3 was phosphorylated on Ser526 in response to osmotic stress. In addition, phosphorylation of Ser526 was required for MKK6 phosphorylation in vitro, whereas dephosphorylation of Ser526 was mediated by protein phosphatase 2A and sensitive to okadaic acid and sodium fluoride. Finally, the association between MEKK3 and 14-3-3 was dependent on Ser526 and prevented dephosphorylation of Ser526. In summary, Ser526 of MEKK3 is an autophosphorylation site within the T-loop that is regulated by PP2A and 14-3-3 proteins.     

The serine 814 of TRPC6 is phosphorylated under unstimulated conditions

TRPC are nonselective cation channels involved in calcium entry. Their regulation by phosphorylation has been shown to modulate their routing and activity. TRPC6 activity increases following phosphorylation by Fyn, and is inhibited by protein kinase G and protein kinase C. A previous study by our group showed that TRPC6 is phosphorylated under unstimulated conditions in a human embryonic kidney cells line (HEK293). To investigate the mechanism responsible for this phosphorylation, we used a MS/MS approach combined with metabolic labeling and showed that the serine at position 814 is phosphorylated in unstimulated cells. The mutation of Ser(814) into Ala decreased basal phosphorylation but did not modify TRPC6 activity. Even though Ser(814) is within a consensus site for casein kinase II (CK2), we showed that CK2 is not involved in the phosphorylation of TRPC6 and does not modify its activity. In summary, we identified a new basal phosphorylation site (Ser(814)) on TRPC6 and showed that CK2 is not responsible for the phosphorylation of this site.     

Differential contribution of troponin I phosphorylation sites to the endothelin-modulated contractile response

Cardiac troponin I is a phosphorylation target for endothelin-activated protein kinase C. Earlier work in cardiac myocytes expressing nonphosphorylatable slow skeletal troponin I provided evidence that protein kinase C-mediated cardiac troponin I phosphorylation accelerates relaxation. However, replacement with the slow skeletal isoform also alters the myofilament pH response and the Ca2+ transient, which could influence endothelin-mediated relaxation. Here, differences in the Ca2+ transient could not explain the divergent relaxation response to endothelin in myocytes expressing cardiac versus slow skeletal troponin I nor could activation of Na+/H+ exchange. Three separate clusters within cardiac troponin I are phosphorylated by protein kinase C, and we set out to determine the contribution of the Thr144 and Ser23/Ser24 clusters to the endothelin-mediated contractile response. Myocyte replacement with a cardiac troponin I containing a Thr144 substituted with the Pro residue found in slow skeletal troponin I resulted in prolonged relaxation in response to acute endothelin compared with control myocytes. Ser23/Ser24 also is a target for protein kinase C phosphorylation of purified cardiac troponin I, and although this cluster was not acutely phosphorylated in intact myocytes, significant phosphorylation developed within 1 h after adding endothelin. Replacement of Ser23/Ser24 with Ala indicated that this cluster contributes significantly to relaxation during more prolonged endothelin stimulation. Overall, results with these mutants provide evidence that Thr144 plays an important role in the acute acceleration of relaxation, whereas Ser23/Ser24 contributes to relaxation during more prolonged activation of protein kinase C by endothelin.     

A region encompassing the FERM domain of Jak1 is necessary for binding to the cytokine receptor gp130

Cardiac L-type Ca(2+) channel is facilitated by protein kinase A (PKA)-mediated phosphorylation. Here, we investigated the role of Ser(1901), a putative phosphorylation site in the carboxy-terminal of rat brain type-II alpha(1C) subunit (rbCII), in the PKA-mediated regulation. Forskolin (3 microM) enhanced Ca(2+) channel currents (I(Ca)) and shifted the activation curve to negative voltages, which were abolished by protein kinase inhibitor. Replacement of Ser(1901) of rbCII by Ala abolished the enhancement of I(Ca) by forskolin but not the shift of the activation curve. These results indicate that Ser(1901) is required for the PKA-mediated enhancement of I(Ca), and that the voltage-dependence of the activation of I(Ca) appears to be modulated via another PKA phosphorylation site.     

Endocytosis of the transferrin receptor requires the cytoplasmic domain but not its phosphorylation site

The transferrin receptor (TR) mediates cellular iron uptake by bringing about the endocytosis of transferrin. We investigated whether the cytoplasmic domain of 65 N-terminal amino acids or phosphorylated sites within this domain constitute a structure that is required for TR endocytosis. To test this hypothesis, we modified the cytoplasmic serine residues or introduced a deletion of 36 amino acids by in vitro mutagenesis of a cDNA expression vector for human TR. Upon expression in transfected mouse Ltk- cells, both the wild-type and phosphorylation site mutant receptors mediated transferrin internalization, whereas the truncated receptor did not. These results provide evidence that the cytoplasmic domain, or part of it, is essential for internalization of the TR, but argue against a role for receptor phosphorylation in endocytosis.     

PKCdelta-mediated IRS-1 Ser24 phosphorylation negatively regulates IRS-1 function

The IRS-1 PH and PTB domains are essential for insulin-stimulated IRS-1 Tyr phosphorylation and insulin signaling, while Ser/Thr phosphorylation of IRS-1 disrupts these signaling events. To investigate consensus PKC phosphorylation sites in the PH-PTB domains of human IRS-1, we changed Ser24, Ser58, and Thr191 to Ala (3A) or Glu (3E), to block or mimic phosphorylation, respectively. The 3A mutant abrogated the inhibitory effect of PKCdelta on insulin-stimulated IRS-1 Tyr phosphorylation, while reductions in insulin-stimulated IRS-1 Tyr phosphorylation, cellular proliferation, and Akt activation were observed with the 3E mutant. When single Glu mutants were tested, the Ser24 to Glu mutant had the greatest inhibitory effect on insulin-stimulated IRS-1 Tyr phosphorylation. PKCdelta-mediated IRS-1 Ser24 phosphorylation was confirmed in cells with PKCdelta catalytic domain mutants and by an RNAi method. Mechanistic studies revealed that IRS-1 with Ala and Glu point mutations at Ser24 impaired phosphatidylinositol-4,5-bisphosphate binding. In summary, our data are consistent with the hypothesis that Ser24 is a negative regulatory phosphorylation site in IRS-1.     

Insulin receptor substrate 2 and FoxO3a signalling are involved in E-cadherin expression and transforming growth factor-β1-induced repression in kidney epithelial cells

Insulin receptor substrate (IRS) proteins comprise a family of adaptor molecules that integrate extracellular signals from insulin and other ligands to intracellular effectors such as phosphoinositide 3-kinase and mitogen-activated protein kinase. The predominant forms of IRS protein in humans, IRS1 and IRS2, are widely expressed. Despite structural similarities, IRS1 and IRS2 display distinct signalling modalities, and mice lacking these proteins present with distinct phenotypes. Transforming growth factor (TGF)-β1 is the primary cytokine shown to induce epithelial-mesenchymal transition. Recent data have demonstrated a role for IRS1 in TGF-β1-induced epithelial-mesenchymal transition in lung epithelial cells. In the present study, we report data showing that TGF-β1 signals via IRS2 in kidney epithelial cells. Small interfering RNA (siRNA)-mediated targeting of IRS2 increased E-cadherin expression, although it did not alter TGF-β1-mediated E-cadherin repression. Phosphorylation of the downstream target of IRS2/Akt signalling, FoxO3a, was induced on Ser253 and, to a lesser extent, on Thr32. Transfection of FoxO3aThr32Ala mutant for 24?h greatly reduced FoxO3a phosphorylation on Ser253 but over-expression of FoxO3a Ser253Ala did not effect Thr32 phosphorylation, suggesting that a distinct order of phosphorylation of FoxO3a is required for physiological function in cells. Transfection of FoxO3a Ser253Ala mutant partially inhibited TGF-β1-mediated E-cadherin repression at 24?h. Taken together, these data highlight novel roles for IRS2 and FoxO3a in the regulation of kidney epithelial cells by E-cadherin.