Fluorescent-Linked Enzyme Chemoproteomic Strategy (FLECS) for Identifying HSP70 Inhibitors.

Activation of the heat shock response, and in particular upregulation of stress-inducible Hsp70, herein referred to as Hsp70i, in newly transformed cells, appears to protect against protein damaging stimuli, induction of premature oncogene-induced terminal senescence (OIS), and apoptosis, thereby enabling tumor initiation and progression to an aggressive phenotype. Expressed at very low or undetectable levels in normal tissue, the cytoprotective effects of Hsp70i appear to be mediated through its activity as a molecular chaperone allowing proper folding of mutated proteins, and by blocking cell signaling pathways that regulate OIS and apoptosis. Identification of small-molecule inhibitors selective for Hsp70i could provide new therapeutic tools for cancer treatment. However, identification of selective inhibitors of Hsp70i has proven challenging largely because of the affinity of the protein for ATP. Additionally, its chaperone functions do not lend the protein amenable to traditional enzymatic high-throughput screens. Here, we describe the use of fluorescence-linked enzyme chemoproteomic strategy (FLECS) to identify Hsp70i inhibitors. The FLECS assay is a simple binding assay that enables proteins tagged with fluorophors to be rapidly and quantitative screened against small-molecule libraries. We show several case history examples of the methodology that led to the discovery of the Fatty acid synthase inhibitor, FASNALL, the DAPK3 inhibitor HS38, and HS72, an allosteric inhibitor selective for Hsp70i.

Human cytidine triphosphate synthetase 1 interacting proteins.

We investigated the interacting proteins and intracellular localization of CTP synthetase 1 (CTPS1) in mammalian cells. CTPS1 interacted with a GST- peptidyl prolyl isomerase, Pin1 fusion (GST-Pin1) in a Ser 575 (S575) phosphorylation-dependent manner. Immunoprecipitation experiments demonstrated that CTPS1 also bound tubulin, and thirteen additional coimmunoprecipitating proteins were identified by mass spectrometry. Immunolocalization experiments showed that tubulin and CTPS1 colocalized subcellularly. Taxol treatment enhanced this but cotreatment of cells with the CTPS inhibitor, cyclopentenyl cytosine (CPEC), and taxol failed to disrupt the colocalization. Thus, these studies provide novel information on the potential interacting proteins that may regulate CTPS1 function or intracellular localization.

Borg proteins control septin organization and are negatively regulated by Cdc42.

The Cdc42 GTPase binds to numerous effector proteins that control cell polarity, cytoskeletal remodelling and vesicle transport. In many cases the signalling pathways downstream of these effectors are not known. Here we show that the Cdc42 effectors Borg1 to Borg3 bind to septin GTPases. Endogenous septin Cdc10 and Borg3 proteins can be immunoprecipitated together by an anti-Borg3 antibody. The ectopic expression of Borgs disrupts normal septin organization. Cdc42 negatively regulates this effect and inhibits the binding of Borg3 to septins. Borgs are therefore the first known regulators of mammalian septin organization and provide an unexpected link between the septin and Cdc42 GTPases.

Site-specific phosphorylation and point mutations of telokin modulate its Ca2+-desensitizing effect in smooth muscle.

Forskolin and 8-bromoguanosine 3'-5'-cyclic monophosphate (8-Br-cGMP) induce phosphorylation of Ser-13 of telokin and relaxation of smooth muscle at constant calcium. Comparison with the effect of wild type with aspartate (D; to mimic phosphorylation) and alanine (A; non-phosphorylatable) mutants of telokin showed that the S13D mutant was more effective than wild type in relaxing smooth muscle at constant calcium. The efficacy of the Ser-13A, S12A, and S12D mutants was not significantly different from that of wild-type telokin. The effect of neither S13D nor Ser-13A was affected by 8-Br-cGMP, whereas the effect of wild type, S12A, and S12D was enhanced by 8-Br-cGMP, indicating the specificity of Ser-13 charge modification. Mutation of Ser-19 (a mitogen-activated protein kinase site) showed the S19A to be more effective than, and S19D to be not different from, wild-type telokin. The effect of both mutants was slightly enhanced by 8-Br-cGMP. A truncated (residues 1-142) form lacking the acidic C terminus had the same relaxant effect as wild-type telokin, whereas the C-terminal peptide (residues 142-155) had no effect. We conclude that site-specific modification of the N terminus modulates the Ca2+ -desensitizing effect of telokin on force.

Dual Ser and Thr phosphorylation of CPI-17, an inhibitor of myosin phosphatase, by MYPT-associated kinase.

Phosphorylation of CPI-17 and PHI-1 by the MYPT1-associated kinase (M110 kinase) was investigated. M110 kinase is a recently identified serine/threonine kinase with a catalytic domain that is homologous to that of ZIP kinase (ZIPK. GST-rN-ZIPK, a constitutively active GST fusion fragment, phosphorylates CPI-17 (but not PHI-1) to a stoichiometry of 1.7 mol/mol. Phosphoamino acid analysis revealed phosphorylation of both Ser and Thr residues. Phosphorylation sites in CPI-17 were identified as Thr 38 and Ser 12 using Edman sequencing with (32)P release and a point mutant of Thr 38.

A role for the Ppz Ser/Thr protein phosphatases in the regulation of translation elongation factor 1Balpha.

In vivo 32P-labeled yeast proteins from wild type and ppz1 ppz2 phosphatase mutants were resolved by bidimensional electrophoresis. A prominent phosphoprotein, which in ppz mutants showed a marked shift to acidic regions, was identified by mixed peptide sequencing as the translation elongation factor 1Balpha (formerly eEF1beta). An equivalent shift was detected in cells overexpressing HAL3, a inhibitory regulatory subunit of Ppz1. Subsequent analysis identified the conserved Ser-86 as the in vivo phosphorylatable residue and showed that its phosphorylation was increased in ppz cells. Pull-down experiments using a glutathione S-transferase (GST)-EF1Balpha fusion version allowed to identify Ppz1 as an in vivo interacting protein. Cells lacking Ppz display a higher tolerance to known translation inhibitors, such as hygromycin and paromomycin, and enhanced readthrough at all three nonsense codons, suggesting that translational fidelity might be affected. Overexpression of a GST-EF1Balpha fusion counteracted the growth defect associated to high levels of Ppz1 and this effect was essentially lost when the phosphorylatable Ser-86 is replaced by Ala. Therefore, the Ppz phosphatases appear to regulate the phosphorylation state of EF1Balpha in yeast, and this may result in modification of the translational accuracy.

Identification of the endogenous smooth muscle myosin phosphatase-associated kinase.

Ca(2+) sensitization of smooth muscle contraction involves inhibition of myosin light chain phosphatase (SMPP-1M) and enhanced myosin light chain phosphorylation. Inhibition of SMPP-1M is modulated through phosphorylation of the myosin targeting subunit (MYPT1) by either Rho-associated kinase (ROK) or an unknown SMPP-1M-associated kinase. Activated ROK is predominantly membrane-associated and its putative substrate, SMPP-1M, is mainly myofibrillar-associated. This raises a conundrum about the mechanism of interaction between these enzymes. We present ZIP-like kinase, identified by "mixed-peptide" Edman sequencing after affinity purification, as the previously unidentified SMPP-1M-associated kinase. ZIP-like kinase was shown to associate with MYPT1 and phosphorylate the inhibitory site in intact smooth muscle. Phosphorylation of ZIP-like kinase was associated with an increase in kinase activity during carbachol stimulation, suggesting that the enzyme may be a terminal member of a Ca(2+) sensitizing kinase cascade.

Phosphorylation of telokin by cyclic nucleotide kinases and the identification of in vivo phosphorylation sites in smooth muscle.

The Ca(2+)-independent acceleration of dephosphorylation of the regulatory light chain of smooth muscle myosin and relaxation of smooth muscle by telokin are enhanced by cyclic nucleotide-activated protein kinase(s) [Wu et al. (1998) J. Biol. Chem. 273, 11362-113691. The purpose of this study was to determine the in vivo site(s) and in vitro rates of telokin phosphorylation and to evaluate the possible effects of sequential phosphorylation by different kinases. The in vivo site(s) of phosphorylation of telokin were determined in rabbit smooth muscles of longitudinal ileum and portal vein. Following stimulation of ileum with forskolin (20 microM) the serine at position 13 was the only amino acid to exhibit increased phosphorylation. Rabbit portal vein telokin was phosphorylated on both Ser-13 and -19 as a result of forskolin and GTPgammaS stimulation in vivo. Point mutation of Ser-13 (to Ala or Asp) abolished in vitro phosphorylation by cyclic nucleotide-dependent protein kinases.

Multiple mechanisms control phosphorylation of PHAS-I in five (S/T)P sites that govern translational repression.

Control of the translational repressor, PHAS-I, was investigated by expressing proteins with Ser/Thr --> Ala mutations in the five (S/T)P phosphorylation sites. Results of experiments with HEK293 cells reveal at least three levels of control. At one extreme is nonregulated phosphorylation, exemplified by constitutive phosphorylation of Ser82. At an intermediate level, amino acids and insulin stimulate the phosphorylation of Thr36, Thr45, and Thr69 via mTOR-dependent processes that function independently of other sites in PHAS-I. At the third level, the extent of phosphorylation of one site modulates the phosphorylation of another. This control is represented by Ser64 phosphorylation, which depends on the phosphorylation of all three TP sites. The five sites have different influences on the electrophoretic properties of PHAS-I and on the affinity of PHAS-I for eukaryotic initiation factor 4E (eIF4E). Phosphorylation of Thr45 or Ser64 results in the most dramatic decreases in eIF4E binding in vitro. However, each of the sites influences mRNA translation, either directly by modulating the binding affinity of PHAS-I and eIF4E or indirectly by affecting the phosphorylation of other sites.

Neurofilament-L is a protein phosphatase-1-binding protein associated with neuronal plasma membrane and post-synaptic density.

Far Westerns with digoxigenin-conjugated protein phosphatase-1 (PP1) catalytic subunit identified PP1-binding proteins in extracts from bovine, rat, and human brain. A major 70-kDa PP1-binding protein was purified from bovine brain cortex plasma membranes, using affinity chromatography on the immobilized phosphatase inhibitor, microcystin-LR. Mixed peptide sequencing following cyanogen bromide digestion identified the 70-kDa membrane-bound PP1-binding protein as bovine neurofilament-L (NF-L). NF-L was the major PP1-binding protein in purified preparations of bovine spinal cord neurofilaments and the cytoskeletal compartment known as post-synaptic density, purified from rat brain cortex. Bovine neurofilaments, at nanomolar concentrations, inhibited the phosphorylase phosphatase activity of rabbit skeletal muscle PP1 catalytic subunit but not the activity of PP2A, another major serine/threonine phosphatase. PP1 binding to bovine NF-L was mapped to the head region. This was confirmed by both binding and inhibition of PP1 by recombinant human NF-L fragments. Together, these studies indicate that NF-L fulfills many of the biochemical criteria established for a PP1-targeting subunit and suggest that NF-L may target the functions of PP1 in membranes and cytoskeleton of mammalian neurons.

Regulatory interactions between the Reg1-Glc7 protein phosphatase and the Snf1 protein kinase.

Protein phosphatase 1, comprising the regulatory subunit Reg1 and the catalytic subunit Glc7, has a role in glucose repression in Saccharomyces cerevisiae. Previous studies showed that Reg1 regulates the Snf1 protein kinase in response to glucose. Here, we explore the functional relationships between Reg1, Glc7, and Snf1. We show that different sequences of Reg1 interact with Glc7 and Snf1. We use a mutant Reg1 altered in the Glc7-binding motif to demonstrate that Reg1 facilitates the return of the activated Snf1 kinase complex to the autoinhibited state by targeting Glc7 to the complex. Genetic evidence indicated that the catalytic activity of Snf1 negatively regulates its interaction with Reg1. We show that Reg1 is phosphorylated in response to glucose limitation and that this phosphorylation requires Snf1; moreover, Reg1 is dephosphorylated by Glc7 when glucose is added. Finally, we show that hexokinase PII (Hxk2) has a role in regulating the phosphorylation state of Reg1, which may account for the effect of Hxk2 on Snf1 function. These findings suggest that the phosphorylation of Reg1 by Snf1 is required for the release of Reg1-Glc7 from the kinase complex and also stimulates the activity of Glc7 in promoting closure of the complex.

Reg1p targets protein phosphatase 1 to dephosphorylate hexokinase II in Saccharomyces cerevisiae: characterizing the effects of a phosphatase subunit on the yeast proteome.

Protein phosphatase 1 (Glc7p) and its binding protein Reg1p are essential for the regulation of glucose repression pathways in Saccharomyces cerevisiae. In order to identify physiological substrates for the Glc7p-Reg1p complex, we examined the effects of deletion of the REG1 gene on the yeast phosphoproteome. Analysis by two-dimensional phosphoprotein mapping identified two distinct proteins that were greatly increased in phosphate content in reg1Delta mutants. Mixed peptide sequencing identified these proteins as hexokinase II (Hxk2p) and the E1alpha subunit of pyruvate dehydrogenase. Consistent with increased phosphorylation of Hxk2p in response to REG1 deletion, fractionation of yeast extracts by anion-exchange chromatography identified Hxk2p phosphatase activity in wild-type strains that was selectively lost in the reg1Delta mutant. The phosphorylation state of Hxk2p and Hxk2p phosphatase activity was restored to wild-type levels in the reg1Delta mutant by expression of a LexA-Reg1p fusion protein. In contrast, expression of LexA-Reg1p containing mutations at phenylalanine in the putative PP-1C-binding site motif (K/R)(X)(I/V)XF was unable to rescue Hxk2p dephosphorylation in intact yeast or restore Hxk2p phosphatase activity. These results demonstrate that Reg1p targets PP-1C to dephosphorylate Hxk2p in vivo and that the motif (K/R)(X) (I/V)XF is necessary for its PP-1 targeting function.

Phosphorylation of the rRNA transcription factor upstream binding factor promotes its association with TATA binding protein.

rRNA synthesis by RNA polymerase I requires both the promoter selectivity factor 1, which is composed of TATA binding protein (TBP) and three TBP-associated factors, and the activator upstream binding factor (UBF). Whereas there is strong evidence implicating a role for phosphorylation of UBF in the control of growth-induced increases in rRNA transcription, the mechanism of this effect is not known. Results of immunoprecipitation studies with TBP antibodies showed increased recovery of phosphorylated UBF from growth-stimulated smooth muscle cells. Moreover, using an immobilized protein-binding assay, we found that phosphorylation of UBF in vivo in response to stimulation with different growth factors or in vitro with smooth muscle cell nuclear extract increased its binding to TBP. Finally, we demonstrated that UBF-TBP binding depended on the C-terminal 'acidic tail' of UBF that was hyperphosphorylated at multiple serine sites after growth factor stimulation. Results of these studies suggest that phosphorylation of UBF and subsequent binding to TBP represent a key regulatory step in control of growth-induced increases in rRNA synthesis.

Phosphorylation of the translational regulator, PHAS-I, by protein kinase CK2.

The primary site in PHAS-I for phosphorylation by protein kinase CK2 in vitro was identified as Ser111. A relatively small amount of phosphorylation of Ser99 was also detected, and mutating Ser99 to Ala in PHAS-I slightly decreased phosphorylation by CK2 in vitro. In contrast, mutating Ser111 to Ala almost abolished phosphorylation, confirming Ser111 as the preferred site for CK2. Phosphorylation of Ser111 did not decrease binding of PHAS-I to eIF4E, and results of peptide mapping experiments with PHAS-I immunoprecipitated from 32P-labeled adipocytes indicated that Ser111 was not phosphorylated in cells. These results support the conclusion that CK2 is not involved in the control of PHAS-I.

Rapid identification of protein phosphatase 1-binding proteins by mixed peptide sequencing and data base searching. Characterization of a novel holoenzymic form of protein phosphatase 1.

Microcystin-affinity chromatography was used to purify 15 protein phosphatase 1 (PP1)-binding proteins from the myofibrillar fraction of rabbit skeletal muscle. To reduce the time and amount of material required to identify these proteins, proteome analysis by mixed peptide sequencing was developed. Proteins are resolved by SDS-polyacrylamide gel electrophoresis, electroblotted to polyvinylidene fluoride membrane, and stained. Bands are sliced from the membrane, cleaved briefly with CnBr, and applied without further purification to an automated Edman sequencer. The mixed peptide sequences generated are sorted and matched against the GenBank using two new programs, FASTF and TFASTF. This technology offers a simple alternative to mass spectrometry for the subpicomolar identification of proteins in polyacrylamide gels. Using this technology, all 15 proteins recovered in PP-1C affinity chromatography were sequenced. One of the proteins, PP-1bp55, was homologous to human myosin phosphatase, MYPT2. A second, PP-1bp80, identified in the EST data bases, contained a putative PP-1C binding site and a nucleotide binding motif. Further affinity purification over ATP-Sepharose isolated PP-1bp80 in a quaternary complex with PP-1C and two other proteins, PP-1bp29 and human p20. Recombinant PP-1bp80 also bound PP-1C and suppressed its activity toward a variety of substrates, suggesting that the protein is a novel regulatory subunit of PP-1.

Acceleration of myosin light chain dephosphorylation and relaxation of smooth muscle by telokin. Synergism with cyclic nucleotide-activated kinase.

Incorporation of 32P into telokin, a smooth muscle-specific, 17-18-kDa, acidic (pI 4.2-4.4) protein, was increased by forskolin (20 microM) in intact rabbit ileum smooth muscle (ileum) and by 8-bromo-cyclic GMP (100 microM) in alpha-toxin-permeabilized ileum. Native telokin (5-20 microM), purified from turkey gizzard, and recombinant rabbit telokin, expressed in Escherichia coli and purified to >90% purity, induced dose-dependent relaxation, associated with a significant decrease in regulatory myosin light chain phosphorylation, without affecting the rate of thiophosphorylation of regulatory myosin light chain of ileum permeabilized with 0.1% Triton X-100. Endogenous telokin was lost from ileum during prolonged permeabilization (>20 min) with 0.1% Triton X-100, and the time course of loss was correlated with the loss of 8-bromo-cyclic GMP-induced calcium desensitization. Recombinant and native gizzard telokins were phosphorylated, in vitro, by the catalytic subunit of cAMP-dependent protein kinase, cGMP-dependent protein kinase, and p42/44 mitogen-activated protein kinase; the recombinant protein was also phosphorylated by calmodulin-dependent protein kinase II. Exogenous cGMP-dependent protein kinase (0.5 microM) activated by 8-bromo-cyclic GMP (50 microM) phosphorylated recombinant telokin (10 microM) when added concurrently to ileum depleted of its endogenous telokin, and their relaxant effects were mutually potentiated. Forskolin (20 microM) also increased phosphorylation of telokin in intact ileum. We conclude that telokin induces calcium desensitization in smooth muscle by enhancing myosin light chain phosphatase activity, and cGMP- and/or cAMP-dependent phosphorylation of telokin up-regulates its relaxant effect.

The amino-terminal domain of heat shock protein 90 (hsp90) that binds geldanamycin is an ATP/ADP switch domain that regulates hsp90 conformation.

Many functions of the chaperone, heat shock protein 90 (hsp90), are inhibited by the drug geldanamycin that specifically binds hsp90. We have studied an amino-terminal domain of hsp90 whose crystal structure has recently been solved and determined to contain a geldanamycin-binding site. We demonstrate that, in solution, drug binding is exclusive to this domain. This domain also binds ATP linked to Sepharose through the gamma-phosphate. Binding is specific for ATP and ADP and is inhibited by geldanamycin. Mutation of four glycine residues within two proposed ATP binding motifs diminishes both geldanamycin binding and the ATP-dependent conversion of hsp90 to a conformation capable of binding the co-chaperone p23. Since p23 binding requires regions outside the 1-221 domain of hsp90, these results indicate a common site for nucleotides and geldanamycin that regulates the conformation of other hsp90 domains.

Identification of phosphorylation sites in the translational regulator, PHAS-I, that are controlled by insulin and rapamycin in rat adipocytes.

Phosphorylation of PHAS-I by mitogen-activated protein (MAP) kinase in vitro decreased PHAS-I binding to eukaryotic initiation factor (eIF)-4E. The decrease in binding lagged behind the phosphorylation of PHAS-I in Ser64, the preferred site of MAP kinase. Binding of the Ala64 mutant of PHAS-I to eIF-4E was abolished by MAP kinase, indicating that phosphorylation of sites other than Ser64 control binding. To identify such sites, PHAS-I was phosphorylated with MAP kinase and [gamma-32P]ATP and then cleaved proteolytically before the resulting phosphopeptides were isolated by reverse phase chromatography and directly identified by amino acid sequencing. Phosphorylated residues were located by determining the cycles in which 32P was released when phosphopeptides were subjected to sequential Edman degradation. With an extended incubation in vitro, MAP kinase phosphorylated Thr36, Thr45, Ser64, Thr69, and Ser82. In rat adipocytes, the phosphorylation of all five sites was increased by insulin and decreased by rapamycin although there were differences in the magnitude of the effects. A form of PHAS-I phosphorylated exclusively in Thr36 remained bound to eIF-4E, indicating that phosphorylation of Thr36 is insufficient for dissociation of the PHAS-I.eIF-4E complex. In summary, our results indicate that multiple phosphorylation sites are involved in the control of PHAS-I. All five sites identified fit a (Ser/Thr)-Pro motif, suggesting that the phosphorylation of PHAS-I in cells is mediated by a proline-directed protein kinase.

Endothelial cell-conditioned medium downregulates smooth muscle contractile protein expression.

Smooth muscle cells (SMC) within atherosclerotic lesions proliferate and exhibit phenotypic modulation, but the contribution of vascular endothelium to this process is poorly understood. Our aim was to examine the effects of endothelial cell-conditioned medium (ECCM) on vascular SMC growth and differentiation. Rat aortic ECCM stimulated a ninefold increase in [3H]thymidine incorporation and downregulated smooth muscle-specific myosin heavy chain and alpha-actin synthesis in rat aortic SMC. These effects were not inhibited by antibodies to platelet-derived growth factor (PDGF)-BB or PDGF-AB or with a PDGF beta-receptor subunit. Treatment with PDGF-BB (at a concentration found in ECCM), PDGF-AA, basic fibroblast growth factor, endothelin-1, or transforming growth factor-beta did not reproduce these effects. The ECCM activities were sensitive to heat and trypsinization, were >30 kDa in molecular mass, and bound weakly to heparin-Sepharose. Our data indicate that cultured endothelial cells produce a factor(s) that downregulates contractile protein expression in SMC, which may contribute to SMC dedifferentiation and proliferation.

PHAS proteins as mediators of the actions of insulin, growth factors and cAMP on protein synthesis and cell proliferation.

PHAS-I and PHAS-II are members of a newly discovered family of proteins that regulate translation initiation. PHAS-I is expressed in a wide variety of cell types, but it is highest in adipocytes, where protein synthesis is markedly increased by insulin. PHAS-II is highest in liver and kidney, where very little PHAS-I is found. PHAS proteins bind to eIF-4E, the mRNA cap-binding protein, and inhibit translation of capped mRNA in vitro and in cells. In rat adipocytes PHAS-I is phosphorylated in at least five sites, all of which conform to the consensus, (Ser/Thr)-Pro. Both PHAS proteins are phosphorylated in response to insulin or growth factors, such as EGF, PDGF and IGF-1. Phosphorylation in the appropriate site(s) promotes dissociation of PHAS/eIF-4E complexes. This allows eIF-4E to bind to eIF-4G (p220), thereby increasing the amount of the eIF-4F complex and the rate of translation initiation. Increasing cAMP promotes PHAS-I dephosphorylation and increases binding to eIF-4E. Unlike PHAS-I, PHAS-II is readily phosphorylated by PKA in vitro, suggesting that regulation of the two proteins differs. However, increasing cAMP in cells also promotes dephosphorylation of PHAS-II. Thus, PHAS proteins appear to be key mediators not only of the stimulatory effects of insulin and growth factors on protein synthesis, but also of the inhibitory effects of cAMP. Moreover, by controlling eIF-4E PHAS proteins may be involved in the control of cell proliferation, as increasing eIF-4E is mitogenic and can even cause malignant transformation of cells. MAP kinase readily phosphorylates both PHAS-I and PHAS-II in vitro, but inhibiting activation of MAP kinase does not attenuate the effects of insulin on increasing phosphorylation of the PHAS proteins in adipocytes or skeletal muscle. MAP kinase phosphorylates neither PHAS-I nor PHAS-II at a significant rate when the proteins are bound to eIF-4E. Therefore, the role of MAP kinase in promoting the dissociation of PHAS/eIF-4E complexes is not clear. Of several protein kinases tested, only casein kinase-II phosphorylated PHAS-I when it was bound eIF-4E. Indeed, the bound form of PHAS-I was phosphorylated more rapidly than the free form. However, it is unlikely that casein kinase II regulates either PHAS protein, as the major site (Ser111) in PHAS-I phosphorylated by casein kinase II in vitro is not phosphorylated in adipocytes, and PHAS-II is not a substrate for casein kinase-II. Pharmacological and genetic evidence indicates that the mTOR/p70S6K pathway is involved in the control of PHAS-I and -II. Thus, PHAS proteins may be mediators of the effects of this pathway on protein synthesis and cell proliferation.