Pinning down proline-directed phosphorylation signaling

The reversible phosphorylation of proteins on serine or threonine residues preceding proline (Ser/Thr-Pro) is a major cellular signaling mechanism. Although it is proposed that phosphorylation regulates the function of proteins by inducing a conformational change, there are few clues about the actual conformational changes and their importance. Recent identification of the novel prolyl isomerase Pin1 that specifically isomerizes only the phosphorylated Ser/Thr-Pro bonds in certain proteins led us to propose a new signaling mechanism, whereby prolyl isomerization catalytically induces conformational changes in proteins following phosphorylation to regulate protein function. Emerging data indicate that such conformational changes have profound effects on catalytic activity, dephosphorylation, protein-protein interactions, subcellular location and/or turnover. Furthermore, this post-phosphorylation mechanism might play an important role in cell growth control and diseases such as cancer and Alzheimer's.     

Bcl-2 Phosphorylation by p38 MAPK: identification of target sites and biologic consequences

The antiapoptotic role of Bcl-2 can be regulated by its phosphorylation in serine and threonine residues located in a nonstructured loop that links BH3 and BH4 domains. p38 MAPK has been identified as one of the kinases able to mediate such phosphorylation, through direct interaction with Bcl-2 protein in the mitochondrial compartment. In this study, we identify, by using mass spectrometry techniques and specific anti-phosphopeptide antibodies, Ser(87) and Thr(56) as the Bcl-2 residues phosphorylated by p38 MAPK and show that phosphorylation of these residues is always associated with a decrease in the antiapoptotic potential of Bcl-2 protein. Furthermore, we obtained evidence that p38 MAPK-induced Bcl-2 phosphorylation plays a key role in the early events following serum deprivation in embryonic fibroblasts. Both cytochrome c release and caspase activation triggered by p38 MAPK activation and Bcl-2 phosphorylation are absent in embryonic fibroblasts from p38alpha knock-out mice (p38alpha(-/-) MEF), whereas they occur within 12 h of serum withdrawal in p38alpha(+/+) MEF; moreover, they can be prevented by p38 MAPK inhibitors and are not associated with the synthesis of the proapoptotic proteins Bax and Fas. Thus, Bcl-2 phosphorylation by activated p38 MAPK is a key event in the early induction of apoptosis under conditions of cellular stress.     

Apoptosis-linked changes in the phosphorylation status and subcellular localization of the spliceosomal autoantigen U1-70K

Apoptosis consists of highly regulated pathways involving post-translational modifications and cleavage of proteins leading to sequential inactivation of the main cellular processes. Here, we focused on the apoptotic processing of one of the essential components of the mRNA splicing machinery, the U1-70K snRNP protein. We found that at an early stage of apoptosis, before the cleavage of the C-terminal part of the protein by caspase-3, the basal phosphorylation of the Ser140 residue located within the RNA recognition motif, increases very significantly. A caspase-dependent, PP1-mediated dephosphorylation of other serine residues takes place in a subset of U1-70K proteins. The U1-70K protein phosphorylated at Ser140 is clustered in heterogeneous ectopic RNP-derived structures, which are finally extruded in apoptotic bodies. The elaborate processing of the spliceosomal U1-70K protein we identified might play an important role in the regulated breakdown of the mRNA splicing machinery during early apoptosis. In addition, these specific changes in the phosphorylation/dephosphorylation balance and the subcellular localization of the U1-70K protein might explain why the region encompassing the Ser140 residue becomes a central autoantigen during the autoimmune disease systemic lupus erythematosus.     

Proline-directed phosphorylation and isomerization in mitotic regulation and in Alzheimer's Disease

The reversible phosphorylation of proteins on serine/threonine residues preceding proline (Ser/Thr-Pro) is a major regulatory mechanism for the control of a series of cell cycle events. Although phosphorylation is thought to regulate protein function by inducing conformational changes, little is known about most of these conformational changes and their significance. Recent studies indicate that the conformation and function of a subset of these phosphorylated proteins are controlled by the prolyl isomerase Pin1 through isomerization of specific phosphorylated Ser/Thr-Pro bonds. Furthermore, compelling evidence supports the idea that proline-directed phosphorylation and subsequent isomerization play a critical role not only in cell cycle control, but also in the development of Alzheimer's disease, where postmitotic neurons display various cell cycle markers, especially mitotic events, prior to degeneration.     

Mimicking phosphorylation of alphaB-crystallin affects its chaperone activity

AlphaB-crystallin is a member of the sHsp (small heat-shock protein) family that prevents misfolded target proteins from aggregating and precipitating. Phosphorylation at three serine residues (Ser19, Ser45 and Ser59) is a major post-translational modification that occurs to alphaB-crystallin. In the present study, we produced recombinant proteins designed to mimic phosphorylation of alphaB-crystallin by incorporating a negative charge at these sites. We employed these mimics to undertake a mechanistic and structural investigation of the effect of phosphorylation on the chaperone activity of alphaB-crystallin to protect against two types of protein misfolding, i.e. amorphous aggregation and amyloid fibril assembly. We show that mimicking phosphorylation of alphaB-crystallin results in more efficient chaperone activity against both heat-induced and reduction-induced amorphous aggregation of target proteins. Mimick-ing phosphorylation increased the chaperone activity of alphaB-crystallin against one amyloid-forming target protein (kappa-casein), but decreased it against another (ccbeta-Trp peptide). We observed that both target protein identity and solution (buffer) conditions are critical factors in determining the relative chaperone ability of wild-type and phosphorylated alphaB-crystallins. The present study provides evidence for the regulation of the chaperone activity of alphaB-crystallin by phosphorylation and indicates that this may play an important role in alleviating the pathogenic effects associated with protein conformational diseases.     

Regulation of insulin receptor substrate 1 pleckstrin homology domain by protein kinase C: role of serine 24 phosphorylation

Phosphorylation of insulin receptor substrate (IRS) proteins on serine residues is an important posttranslational modification that is linked to insulin resistance. Several phosphoserine sites on IRS1 have been identified; the majority are located proximal to the phosphotryosine-binding domain or near key receptor tyrosine kinase substrate- and/or Src-homology 2 domain-binding sites. Here we report on the characterization of a serine phosphorylation site in the N-terminal pleckstrin homology (PH) domain of IRS1. Bioinformatic tools identify serine 24 (Ser24) as a putative substrate site for the protein kinase C (PKC) family of serine kinases. We demonstrate that this site is indeed a bona fide substrate for conventional PKC. In vivo, IRS-1 is also phosphorylated on Ser24 after phorbol 12-myristate 13-acetate treatment of cells, and isoform-selective inhibitor studies suggest the involvement of PKCalpha. By comparing the pharmacological characteristics of phorbol 12-myristate 13-acetate-stimulated Ser24 phosphorylation with phosphorylation at two other sites previously linked to PKC activity (Ser307 and Ser612), we show that PKCalpha is likely to be directly involved in Ser24 phosphorylation, but indirectly involved in Ser307 and Ser612 phosphorylation. Using Ser24Asp IRS-1 mutants to mimic the phosphorylated residue, we demonstrate that the phosphorylation status of Ser24 does play an important role in regulating phosphoinositide binding to, and the intracellular localization of, the IRS1-PH domain, which can ultimately impinge on insulin-stimulated glucose uptake. Hence we provide evidence that IRS1-PH domain function is important for normal insulin signaling and is regulated by serine phosphorylation in a manner that could contribute to insulin resistance.     

Identification of phosphorylation sites in mammalian mitochondrial ribosomal protein DAP3

Mammalian mitochondrial ribosomes synthesize 13 proteins that are essential for oxidative phosphorylation. In addition to their role in protein synthesis, some of the mitochondrial ribosomal proteins have acquired functions in other cellular processes such as apoptosis. Death-associated protein 3 (DAP3), also referred to as mitochondrial ribosomal protein S29 (MRP-S29), is a GTP-binding pro-apoptotic protein located in the small subunit of the ribosome. Previous studies have shown that phosphorylation is one of the most likely regulatory mechanisms for DAP3 function in apoptosis and may be in protein synthesis; however, no phosphorylation sites were identified. In this study, we have investigated the phosphorylation status of ribosomal DAP3 and mapped the phosphorylation sites by tandem mass spectrometry. Mitochondrial ribosomal DAP3 is phosphorylated at Ser215 or Thr216, Ser220, Ser251 or Ser252, and Ser280. In addition, phosphorylation of recombinant DAP3 by Protein kinase A and Protein kinase Cdelta at residues that are endogenously phosphorylated in ribosomal DAP3 suggests both of these kinases as potential candidates responsible for the in vivo phosphorylation of DAP3 in mammalian mitochondria. Interestingly, the majority of the phosphorylation sites detected in our study are clustered around the highly conserved GTP-binding motifs, speculating on the significance of these residues on protein conformation and activity. Site-directed mutagenesis studies on selected phosphorylation sites were performed to determine the effect of phosphorylation on cell proliferation and PARP cleavage as indication of caspase activation. Overall, our findings suggest DAP3, a mitochondrial ribosomal small subunit protein, is a novel phosphorylated target.     

Conserved phosphorylation of serines in the Ser-X-Glu/Ser(P) sequences of the vitamin K-dependent matrix Gla protein from shark, lamb, rat, cow, and human.

The present studies demonstrate that matrix Gla protein (MGP), a 10-kDa vitamin K-dependent protein, is phosphorylated at 3 serine residues near its N-terminus. Phosphoserine was identified at residues 3, 6, and 9 of bovine, human, rat, and lamb MGP by N-terminal protein sequencing. All 3 modified serines are in tandemly repeated Ser-X-Glu sequences. Two of the serines phosphorylated in shark MGP, residues 2 and 5, also have glutamate residues in the n + 2 position in tandemly repeated Ser-X-Glu sequences, whereas the third, shark residue 3, would acquire an acidic phosphoserine in the n + 2 position upon phosphorylation of serine 5. The recognition motif found for MGP phosphorylation, Ser-X-Glu/Ser(P), has been seen previously in milk caseins, salivary proteins, and a number of regulatory peptides. A review of the literature has revealed an intriguing dichotomy in the extent of serine phosphorylation among secreted proteins that are phosphorylated at Ser-X-Glu/Ser(P) sequences. Those phosphoproteins secreted into milk or saliva are fully phosphorylated at each target serine, whereas phosphoproteins secreted into the extracellular environment of cells are partially phosphorylated at target serine residues, as we show here for MGP and others have shown for regulatory peptides and the insulin-like growth factor binding protein 1. We propose that the extent of serine phosphorylation regulates the activity of proteins secreted into the extracellular environment of cells, and that partial phosphorylation can therefore be explained by the need to ensure that the phosphoprotein be poised to gain or lose activity with regulated changes in phosphorylation status.(ABSTRACT TRUNCATED AT 250 WORDS)     

Location and function of three sites phosphorylated on rat acetyl-CoA carboxylase by the AMP-activated protein kinase

1. We have sequenced two tryptic/chymotryptic peptides (TC3 and TC3a) containing a third site phosphorylated on rat acetyl-CoA carboxylase by the AMP-activated protein kinase. Comparison with the complete sequence of rat acetyl-CoA carboxylase predicted from the cDNA sequence [López-Casillas et al. (1988) Proc. Natl Acad. Sci. USA 85, 5784-5788] shows that this site corresponds to Ser1215. 2. Comparison of the cDNA sequence with previous amino acid sequence data identifies the other two sites for the AMP-activated protein kinase as Ser79 and Ser1200. A total of eight serine residues phosphorylated in vitro by six protein kinases can now be identified: six of these (Ser23, Ser25, Ser29, Ser77, Ser79 and Ser95) are clustered in the amino terminal region, while two (Ser1200 and Ser1215) are located in the central region. 3. Prior phosphorylation of Ser77 and Ser1200 by cyclic-AMP-dependent protein kinase prevents subsequent phosphorylation of Ser79 and Ser1200, but not Ser1215, by the AMP-activated protein kinase. Phosphorylation of Ser1215 under these conditions is not associated with a change in enzyme activity. 4. Limited trypsin treatment of native acetyl-CoA carboxylase selectively cleaves off the highly phosphorylated amino-terminal region containing Ser79. 5. Phosphorylation at Ser79 and Ser1200 by the AMP-activated protein kinase dramatically decreases Vmax and increases the A0.5 for citrate. Phosphorylation at Ser77 and Ser1200 by cyclic-AMP-dependent protein kinase causes more modest changes in the A0.5 for citrate and the Vmax. Dephosphorylation, or removal of the amino-terminal region containing Ser77/79 using trypsin, reverses all of these effects. 6. These results suggest that the effects of the AMP-activated protein kinase on acetyl-CoA carboxylase activity are mediated entirely by phosphorylation of Ser79, and not Ser1200 and Ser1215. The smaller effects of cyclic-AMP-dependent protein kinase are mediated by phosphorylation of Ser77.     

Pinning down cell signaling, cancer and Alzheimer's disease

Protein phosphorylation on certain serine or threonine residues preceding proline (Ser/Thr-Pro) is a pivitol signaling mechanism in diverse cellular processes and its deregulation can lead to human disease. However, little is known about how these phosphorylation events actually control cell signaling. Pin1 is a highly conserved enzyme that isomerizes only the phosphorylated Ser/Thr-Pro bonds in certain proteins, thereby inducing conformational changes. Recent results indicate that such conformational changes following phosphorylation are a novel signaling mechanism pivotal in regulating many cellular functions. This mechanism also offers new insights into the pathogenesis and treatment of human disease, most notably cancer and Alzheimer's disease. Thus, Pin1 plays a key role in linking signal transduction to the pathogenesis of cancer and Alzheimer's disease - two major age-related diseases.     

Phosphorylation of specific serine residues in the PKR activation domain of PACT is essential for its ability to mediate apoptosis

Activation of the latent protein kinase, PKR, by extracellular stresses and triggering of resultant cellular apoptosis are mediated by the protein, PACT, which itself gets phosphorylated in stressed cells. We have analyzed the underlying biochemical mechanism by carrying out alanine-scanning mutagenesis of the PKR activation domain of PACT. Among the indispensable residues identified were two serine residues, whose phosphorylation was essential for the cellular actions of PACT. Two-dimensional gel analysis, Western analysis using phosphoamino acid-specific antiserum, and in vivo 32P labeling of PACT demonstrated that constitutive phosphorylation of one of the two residues, Ser246, was required for stress-induced phosphorylation of the other, Ser287. Substitution of either of them by threonine or aspartic acid, but not alanine, was tolerated. Substitution of both residues with the phosphoserine mimetic, aspartic acid, produced a mutant PACT that, unlike the wild-type protein, caused PKR activation and apoptosis, even in unstressed cells. These results indicate that phosphorylation of specific serine residues in the activation domain of PACT is the major mode of transmission of cellular stress response to PKR.     

Insulin-induced stimulation of JNK and the PI 3-kinase/mTOR pathway leads to phosphorylation of serine 318 of IRS-1 in C2C12 myotubes

Increased serine/threonine phosphorylation of insulin receptor substrate-1 (IRS-1) is associated with cellular insulin resistance. We have recently identified serine 318 (Ser318) as a novel protein kinase C-zeta (PKC-zeta)-dependent phosphorylation site within IRS-1. As other kinases may phosphorylate at this serine residue as well, we aimed to identify such kinases in the present study. In C2C12 myotubes, exposure to insulin or phorbol ester markedly increased Ser318 phosphorylation. In contrast, high glucose, tumor necrosis factor-alpha, and free fatty acids did not provoke Ser318 phosphorylation. JNK and the PI 3-kinase/mTOR pathway were found to be implicated in insulin-induced Ser318 phosphorylation, but not in TPA-stimulated phosphorylation that was, at least partly, mediated by classical or novel PKC. In conclusion, with JNK and the PI 3-kinase/mTOR pathway as mediators of insulin-induced Ser318 phosphorylation, we have identified kinases that have previously been reported to play key roles in phosphorylation of other serine residues in IRS-1.     

Mono- or double-site phosphorylation distinctly regulates the proapoptotic function of Bax

Bax is the major multidomain proapoptotic molecule that is required for apoptosis. It has been reported that phosphorylation of Bax at serine(S) 163 or S184 activates or inactivates its proapoptotic function, respectively. To uncover the mechanism(s) by which phosphorylation regulates the proapoptotic function of Bax, a series of serine (S)→ alanine/glutamate (A/E) Bax mutants, including S163A, S184A, S163E, S184E, S163E/S184A (EA), S163A/S184E (AE), S163A/S184A (AA) and S163E/S184E (EE), were created to abrogate or mimic, respectively, either single or double-site phosphorylation. The compound Bax mutants (i.e. EA and AE) can flesh out the functional contribution of individual phosphorylation site(s). WT and each of these Bax mutants were overexpressed in Bax(-/-) MEF or lung cancer H157 cells and the proapoptotic activities were compared. Intriguingly, expression of any of Bax mutants containing the mutation S→A at S184 (i.e. S184A, EA or AA) represents more potent proapoptotic activity as compared to WT Bax in association with increased 6A7 epitope conformational change, mitochondrial localization/insertion and prolonged half-life. In contrast, all Bax mutants containing the mutation S→E at S184 (i.e. S184E, AE or EE) have a mobility-shift and fail to insert into mitochondrial membranes with decreased protein stability and less apoptotic activity. Unexpectedly, mutation either S→A or S→E at S163 site does not significantly affect the proapoptotic activity of Bax. These findings indicate that S184 but not S163 is the major phosphorylation site for functional regulation of Bax's activity. Therefore, manipulation of the phosphorylation status of Bax at S184 may represent a novel strategy for cancer treatment.     

Substitution of the autophosphorylation site Thr516 with a negatively charged residue confers constitutive activity to mouse 3-phosphoinositide-dependent protein kinase-1 in cells

3-Phosphoinositide-dependent protein kinase-1 (PDK-1)is a serine/threonine kinase that has been found to phosphorylate and activate several members of the AGC protein kinase family including protein kinase B (Akt), p70 S6 kinase, and protein kinase Czeta. However, the mechanism(s) by which PDK-1 is regulated remains unclear. Here we show that mouse PDK-1 (mPDK-1) undergoes autophosphorylation in vitro on both serine and threonine residues. In addition, we have identified Ser(399) and Thr(516) as the major mPDK-1 autophosphorylation sites in vitro. Furthermore, we have found that these two residues, as well as Ser(244) in the activation loop, are phosphorylated in cells and demonstrated that Ser(244) is a major in vivo phosphorylation site. Abolishment of phosphorylation at Ser(244), but not at Ser(399) or Thr(516), led to a significant decrease of mPDK-1 autophosphorylation and kinase activity in vitro, indicating that autophosphorylation at Ser(399) or Thr(516) is not essential for mPDK-1 autokinase activity. However, overexpression of mPDK-1(T516E), but not of mPDK-1(S244E) or mPDK-1(S399D), in Chinese hamster ovary and HEK293 cells was sufficient to induce Akt phosphorylation at Thr(308) to a level similar to that of insulin stimulation. Furthermore, this increase in phosphorylation was independent of the Pleckstrin homology domain of Akt. Taken together, our results suggest that mPDK-1 undergoes autophosphorylation at multiple sites and that this phosphorylation may be essential for PDK-1 to interact with and phosphorylate its downstream substrates in vivo.     

Vinblastine-induced apoptosis is mediated by discrete alterations in subcellular location, oligomeric structure, and activation status of specific Bcl-2 family members

To gain a broader insight into the role of Bcl-2 proteins in apoptosis induced after mitotic arrest, we investigated the subcellular location, oligomeric structure, and protein interactions of Bax, Bcl-2, and Bcl-xL in vinblastine-treated KB-3 cells. Vinblastine induced the translocation of Bax from the cytosol to the mitochondria, which was accompanied by conformational activation and oligomerization of Bax. Bcl-2 was located in the mitochondria, underwent multisite phosphorylation after vinblastine treatment, and was strictly monomeric under all conditions. In contrast, in control cells, Bcl-xL existed in both monomeric (30 kDa) and oligomeric (150 kDa) forms. Treatment with agents that induced Bcl-xL phosphorylation (microtubule inhibitors) caused loss of the 150-kDa form, but this species was unaffected by apoptotic stimuli that did not stimulate phosphorylation. Vinblastine also promoted Bax activation and Bax oligomerization in HCT116 colon cancer cells. Both wild-type and Bax-deficient HCT116 cells expressed the 150-kDa form of Bcl-xL, which was depleted similarly in both cell lines upon vinblastine treatment. Co-immunoprecipitation studies revealed that in untreated KB-3 cells inactive cytosolic Bax interacted with Bcl-xL, whereas in vinblastine-treated cells, activated mitochondrial Bax did not interact with Bcl-xL. Interaction of Bcl-2 with Bax was not observed under any condition. Overexpression of Bcl-xL inhibited vinblastine-induced Bax activation and Bax dimerization and in parallel inhibited apoptosis. The results indicate that vinblastine-induced apoptosis requires translocation, activation, and oligomerization of Bax and is associated with specific changes in the oligomeric properties of Bcl-xL, which occur independently of Bax.     

Analysis of the role of conserved cysteine residues in the bcl-2 oncoprotein

The Bcl-2 oncoprotein is an integral membrane protein localized primarily to the outer membrane of the mitochondria. The precise molecular mechanism responsible for the antiapoptotic action of Bcl-2 remains unknown. Two cysteine residues are found in Bcl-2 and these residues are well-conserved across species. The first cysteine (cys(155)) is located in the alpha5 domain, a region important for the ion channel properties of Bcl-2, while the second cysteine (cys(226)) is located in the carboxyl-terminal membrane anchor domain. In this study, we found that replacement of both cysteines with serine residues generated a mutant protein that retained the ability to homodimerize and heterodimerize with proapoptotic Bax protein in vitro. In whole cells, the mutant protein efficiently heterodimerized with Bax, but exhibited impaired homodimerizationrelative to wild-type Bcl-2. The mutant protein was also less efficient than wild-type Bcl-2 at suppressing caspase activation, DNA fragmentation, and loss of viability during IL-3 withdrawal-induced apoptosis. Together, the data indicate that the cysteine residues in Bcl-2 contribute, but are not absolutely essential, to the ability of Bcl-2 to homodimerize, heterodimerize with Bax, and suppress apoptosis.