Deciliation Induces Phosphorylation of a 90-kDa Cortical Protein in Tetrahymena thermophila

ABSTRACT. We have used the anti-phosphoprotein antibody MPM-2 to examine changes in phosphorytation of cortical proteins during cilia regeneration in Tetrahymena thermophila . Although numerous cortical proteins are phosphorylated in both nondeciliated and deciliated cells, deciliation induces a dramatic increase in the phosphorylation of a 90-kDa cortical protein. The 90-kDa protein remained phosphorylated during cilia regeneration and then gradually became dephosphorylated. The 90-kDa protein was phosphorylated and dephosphorylated normally in Tetrahymena mutants that assemble short cilia, suggesting that achievement of full length is not the signal that triggers dephosphorylation of the 90-kDa protein. When initiation of cilia assembly is blocked, the 90-kDa protein becomes phosphorylated and remains phosphorylated for an extended period of time, suggesting that initiation of cilia elongation triggers eventual dephosphorylation of the 90-kDa protein, regardless of how long the cilia actually become.     

A monoclonal antibody to animal centrosomes recognizes components of the basal-body root complex inChlamydomonas

Summary The mammalian centrosome monoclonal antibody MPM-13 recognized component(s) of the well defined MTOC basal-body root complex in the green plantChlamydomonas. The antibody reaction coincided in location with the basal-body root complex and the cruciate nature of the staining pattern corresponded to the configuration of the root microtubules. During mitosis the behaviour of MPM-13 stained material mirrored the duplication, separation and migration to the spindle poles of the basal body-root complex. It is suggested that conserved MTOC components were recognized and that these may have retained a similar, perhaps universal, function in microtubule organization.Abbreviations BSA bovine serum albumin - DAPI 4,6-diamidine-2-phenylindole dihydrochloride - mt mating type - MT microtubule - MTOC microtubule organizing centre - PFA paraformaldehyde - PBS phosphate buffered saline     

Unusual phyletic distribution of peptidases as a tool for identifying potential drug targets

Profound changes in the phosphorylation state of many proteins occur during mitosis. It is well established that many of these mitotic phosphorylations are carried out by archetypal mitotic kinases that are activated only during mitosis, shifting the equilibrium of kinases and phosphatases towards phosphorylation. However, many studies have also detailed the phosphorylation of proteins at mitosis by kinases that are constitutively active throughout the cell cycle. In most cases, it is uncertain how kinases and phosphatases that appear to be constitutively active can induce phosphorylations specifically at mitosis. In this issue of the Biochemical Journal, Escargueil and Larsen provide evidence of an interesting alternative mechanism to attain specific mitotic phosphorylation. A mitosis-specific phosphorylation site in DNA topoisomerase IIalpha, which is recognized by the MPM-2 antibody, is phosphorylated by protein kinase CK2. The authors found that phosphorylation of this site is suppressed during interphase due to competing dephosphorylation by protein phosphatase 2A. Interestingly, protein phosphatase 2A is excluded from the nucleus during early mitosis, allowing CK2 to phosphorylate topoisomerase IIalpha. It is possible that similar mechanisms are used to regulate the phosphorylation of other proteins.     

The MPM-2 antibody inhibits mitogen-activated protein kinase activity by binding to an epitope containing phosphothreonine-183.

Mitogen-activated protein (MAP) kinases are a family of serine/threonine kinases implicated in the control of cell proliferation and differentiation. We have found that activated p42mapk is a target for the phosphoepitope antibody MPM-2, a monoclonal antibody that recognizes a cell cycle-regulated phosphoepitope. We have determined that the MPM-2 antibody recognizes the regulatory region of p42mapk. Binding of the MPM-2 antibody to active p42mapk in vitro results in a decrease in p42mapk enzymatic activity. The MPM-2 phosphoepitope can be generated in vitro on bacterially expressed p42mapk by phosphorylation with either isoform of MAP kinase kinase (MKK), MKK1, or MKK2. Analysis of p42mapk proteins mutated in their regulatory sites shows that phosphorylated Thr-183 is essential for the binding of the MPM-2 antibody. MPM-2 binding to Thr-183 is affected by the amino acid present in the other regulatory site, Tyr-185. Substitution of Tyr-185 with phenylalanine results in strong binding of the MPM-2 antibody, whereas substitution with glutamic acid substantially diminishes MPM-2 antibody binding. The MPM-2 phosphoepitope antibody recognizes an amino acid domain incorporating the regulatory phosphothreonine on activated p42mapk in eggs during meiosis and in mammalian cultured cells during the G0 to G1 transition.     

Acquisition of cell polarity during cell cycle and oral replacement in Tetrahymena

The aim of this study was to search for a mechanism responsible for the acquisition of cell polarity in a ciliate Tetrahymena. Homologs of the mammalian genes coding for CDC42-GSK3beta- MARK/PAR1-MAPs proteins were found in the Tetrahymena genome (Eisen et al., 2006, and this study). These proteins belong to a pathway which controls assembly and disassembly of microtubule bundles and cell polarity in neural cells. In Tetrahymena, there are two types of morphogenesis: divisional and oral replacement (OR). In divisional morphogenesis, an elongation of longitudinal microtubule bundles (LMs) takes place during cell division. In contrast, in OR type morphogenesis, which occurs in starved non-dividing cells, a polar retraction of LMs occurs. In T. pyriformis, the frequency of developmental switch to OR morphogenesis increases in the presence of wortmannin, an inhibitor of the CDC42-GSK3beta-MARK pathway. In contrast, wortmannin when applied to dividing cells does not affect divisional morphogenesis. Using immunostaining with the antibody against mammalian mitotic phosphoproteins (MPM-2) we show that these proteins co-localize with the LMs and are distributed along the anterior-posterior gradient. In addition, we show that during OR type morphogenesis, the fate of LMs correlates with the anterior-posterior gradient of instability of the cortical structures. We used the conditional mouth-less mutant of T. thermophila (Tiedtke et al., 1988) to test if the presence of the oral apparatus is required for the maintenance of cell polarity. We discuss our results in relation to the hypothesis of GSK3-beta-MARK pathway involvement in the acquisition of cell polarity in Tetrahymena.     

Threonine phosphorylation is associated with mitosis in HeLa cells

Phosphorylation and dephosphorylation of proteins play an important role in the regulation of mitosis and meiosis. In our previous studies we have described mitosis-specific monoclonal antibody MPM-2 that recognizes a family of phosphopeptides in mitotic cells but not in interphase cells. These peptides are synthesized in S phase but modified by phosphorylation during G2/mitosis transition. The epitope for the MPM-2 is a phosphorylated site. In this study, we attempted to determine which amino acids are phosphorylated during the G2-mitosis (M) transition. We raised a polyclonal antibody against one of the antigens recognized by MPM-2, i.e. a protein of 55 kDa, that is present in interphase cells but modified by phosphorylation during mitosis. This antibody recognizes the p55 protein in both interphase and mitosis while it is recognized by the monoclonal antibody MPM-2 only in mitotic cells. Phosphoamino acid analysis of protein p55 from 32P-labeled S-phase and M-phase HeLa cell extracts after immunoprecipitation with anti-p55 antibodies revealed that threonine was extensively phosphorylated in p55 during G2-M but not in S phase, whereas serine was phosphorylated during both S and M phases. Tyrosine was not phosphorylated. Identical results were obtained when antigens recognized by MPM-2 were subjected to similar analysis. As cells completed mitosis and entered G1 phase phosphothreonine was completely dephosphorylated whereas phosphoserine was not. These results suggest that phosphorylation of threonine might be specific to some of the mitosis-related events.     

Probing microtubule organizing centres with MPM-2 in dividing cells of higher plants using immunofluorescence and immunogold techniques

Summary The localization in higher plant cells of phosphorylated proteins recognized by the monoclonal antibody MPM-2 was investigated, with particular attention to putative microtubule organizing centres (MTOCs). Immunofluorescence and immunogold electron microscopy showed that MPM-2 did not localize with most putative MTOCs in cells and protoplasts of the gymnospermPicea glauca and in cells of the angiospermVicia faba. The distribution of phosphoproteins detected by MPM-2 was similar during mitosis in both species. At late interphase and early prophase MPM-2 preferentially labelled nucleoli and the region around the condensing chromosomes but not the cytoplasm. General labelling of the cytoplasm followed dissolution of the nuclear envelope and by prometaphase centromeres stained strongly. At metaphase and very early anaphase kinetochores stained strongly by immunofluorescence but only weakly using immunogold; spindle microtubules (MTs) showed little staining. Kinetochore staining disappeared during anaphase and by telophase centromeres and loose regions of chromatin in reforming nuclei were labelled. Treatment with the anti-microtubular drug amiprophosmethyl (APM) showed that the phosphorylation/dephosphorylation cycle detected by MPM-2 proceeded independently of the mitotic spindle. Staining of centromeres/kinetochores with MPM-2 suggests that phosphorylation and dephosphorylation of this region of mitotic chromosomes may be involved in chromosome organization, chromatid separation and MT nucleation and/or attachment.Abbreviations APM amiprophos-methyl - DAPI 4,6-diamidino-2-phenylindole - EGTA ethylene glycol-bis(-aminoethyl ether) - FITC fluorescein isothiocyanate - MT microtubule - MTOC microtubule organizing centre - MtSB microtubule stabilizing buffer - PBS phosphate buffered saline - PBSB phosphate buffered saline with bovine serum albumin - PIPES piperazine-N,N-bis (2-ethanesulfonic acid) - PPB preprophase band - SPB spindle pole body - TRITC tetramethylrhodamine isothiocyanate     

Mitotic phosphorylation of DNA topoisomerase II alpha by protein kinase CK2 creates the MPM-2 phosphoepitope on Ser-1469

DNA topoisomerase II alpha is required for chromatin condensation during prophase. This process is temporally linked with the appearance of mitosis-specific phosphorylation sites on topoisomerase IIalpha including one recognized by the MPM-2 monoclonal antibody. We now report that the ability of mitotic extracts to create the MPM-2 epitope on human topoisomerase II alpha is abolished by immunodepletion of protein kinase CK2. Furthermore, the MPM-2 phosphoepitope on topoisomerase II alpha can be generated by purified CK2. Phosphorylation of C-truncated topoisomerase II alpha mutant proteins conclusively shows, that the MPM-2 epitope is present in the last 163 amino acids. Use of peptides containing all conserved CK2 consensus sites in this region indicates that only the peptide containing Arg-1466 to Ala-1485 is able to compete with topoisomerase II alpha for binding of the MPM-2 antibody. Replacement of Ser-1469 with Ala abolishes the ability of the phosphorylated peptide to bind to the MPM-2 antibody while a peptide containing phosphorylated Ser-1469 binds tightly. Surprisingly, the MPM-2 phosphoepitope influences neither the catalytic activity of topoisomerase II alpha nor its ability to form molecular complexes with CK2 in vitro. In conclusion, we have identified protein kinase CK2 as a new MPM-2 kinase able to phosphorylate an important mitotic protein, topoisomerase II alpha, on Ser-1469.     

cdc25 is one of the MPM-2 antigens involved in the activation of maturation-promoting factor.

MPM-2 antigens, a discrete set of phosphoproteins that contain similar phosphoepitopes recognized by the monoclonal antibody MPM-2, are phosphorylated during M-phase induction. Our previous studies suggested that certain MPM-2 antigens are involved in the appearance of maturation-promoting factor (MPF) activity. Because the central mitotic regulator cdc2 kinase has been shown to exhibit MPF activity, we explored the possibility that certain MPM-2 antigens are regulators of cdc2 kinase. We found that MPM-2 binding of its antigens would inhibit the autoamplification of cdc2 kinase in Xenopus oocytes and interfere with cyclin-activation of cdc2 kinase in Xenopus interphase egg extract. Immunodepletion of MPM-2 antigens from cyclin-induced M-phase egg extract caused the inactivation of cdc2 kinase, which was accompanied by an inhibitory phosphorylation of p34cdc2 on Thr 14 and Tyr 15, indicating that at least one MPM-2 antigen is a positive regulator of p34cdc2 dephosphorylation. We then showed that cdc25 from M-phase arrested egg extract is an MPM-2 antigen. These results suggest that phosphorylation of the epitope recognized by MPM-2 may be a crucial event in the activation of cdc25 and that the kinase(s) that phosphorylates this MPM-2 epitope may be an important regulator of cdc2 kinase activation.     

Ribosomal proteins in growing and starved Tetrahymena pyriformis. Starvation-induced phosphorylation of ribosomal proteins.

The complements of ribosomal proteins in growing and starved cells of Tetrahymena pyriformis strain GL were examined by two-dimensional gel electrophoresis. In growing cells, the 40-S ribosomal subunit contained 30 proteins, 4 of which migrated toward the anode at pH 8.6, while the 60-S ribosomal subunit contained 46 proteins, 9 of which migrated toward the anode at pH 8.6. When exponentially growing cells were transferred into a non-nutrient medium pronounced phosphorylation of a single 40-S ribosomal subunit protein, S6, was induced. The phosphorylation was very specific; more than 99.5% of the [32P]phosphate incorporated into ribosomal proteins was associated with S6. Phosphate was incorporated into S6 as O-phosphoserine and O-phosphothreonine. Two-dimensional gel electrophoresis indicated that the complement of proteins associated with the ribosomes isolated from starved cells differed from that of growing cells. Careful examination, however, suggested that except for the phosphorylation of certain ribosomal proteins in starved cells, the observed differences did not reflect starvation-induced changes in vivo, but most probably different levels of artifactual modifications (limited proteolysis) during the preparation of the ribosomes.     

Large-scale identification of novel mitosis-specific phosphoproteins

Systematic identification of phosphoproteins is essential for understanding cellular signalling pathways since phosphorylation plays important roles in cellular regulation. Monoclonal antibody MPM-2 recognizes a discrete set of mitosis-specific phosphoproteins and constitutes a specific tool to investigate the significance of phosphorylation in cell cycle. However, due to the difficulties in identifying antigens revealed on immunoblot membrane, only minority of MPM-2 antigens have been identified. Here we originated proteomics approaches for large-scale identification of MPM-2 phosphoproteins. Mitotic extracts were run on several two-dimensional gel electrophoresis (2D) in parallel, and stained by Coomassie Blue. Each individual spot on one of the gels was excised, and proteins in it were further resolved by regular SDS-electrophoresis and blotted on membrane for MPM-2 stain. Counterparts of the positive proteins were selected on another parallel 2D gel and identified by mass-spectrometry. Using this strategy, 100 spots were excised from Coomassie-stained 2D gel and screened by 1D immunoblots for MPM-2 reactivity, and 22 proteins containing potential MPM-2 epitope were identified in addition to a known MPM-2 antigen, laminin-binding protein. These results were further validated by immunofluorescence, co-immunoprecipitation and in vitro phosphorylation assay. The identification of an unprecedented number of potential MPM-2 phosphoprotein antigens gives new insight into the range of proteins involved in the regulation of the early stages of cell division. Meanwhile, this strategy could be used wherever unknown antigens are explored, especially for antibodies that can recognize more than one antigen.     

RNA synthesis in starved deciliated Tetrahymena pyriformis.

Tetrahymena pyriformis which has been starved for 20 h by incubation in buffer, and then deciliated, can regenerate its cilia in about 90 min while still in suspension in non-nutrient medium. The process of reciliation is accompanied by protein synthesis which begins a few minutes after deciliation and by synthesis of ribosomal and messenger RNAs during a period extending from about 1 h to about 3 h after deciliation. Although net synthesis of RNA remains at a very low level until 1 h after deciliation, a qualitative change in the translatable poly(A)-containing messenger RNA content of deciliated cells, and in particular, formation of beta-tubulin mRNA can be detected almost immediately after deciliation.     

Fine structural studies of the bipolarization of the mitotic apparatus in the fertilized sea urchin egg. I. The structure and behavior of centrosomes before fusion of the pronuclei

During fertilization the sperm brings two centrosomes into the egg. One centrosome contains a centriole of normal length originally seen as the basal body of the sperm flagellum. Characteristically, the proximal half is enwrapped in osmiophilic material. This centrosome is attached to the centrosomal fossa, a bowl-shaped depression of the nuclear envelope of the male pronucleus. Microtubules radiate out from the osmiophilic half characterizing this structure as a centrosome and microtubule organizing center (MTOC). The second centrosome which also acts as an MTOC is attached to the mitochondrion of the sperm. At the beginning it appears as an unstructured accumulation of osmiophilic material out of which later on centriolar microtubules grow. Though this centrosome is marked by an immature centriole it is capable of organizing microtubules and of reproducing itself. This centrosome becomes loosely associated with the female pronucleus by means of microtubules. Then it separates from the mitochondrion which finally is lost. When the two pronuclei fuse, the centrosome derived from the basal body remains firmly attached to the centrosomal fossa, which has persisted in the envelope of the zygote nucleus after pronuclear fusion. Using the fossa as a marker of the position of this centrosome on the nuclear surface, we conclude that it is a stationary centrosome in the process of bipolarization for the first mitosis.     

Protein phosphorylation and dynamics of cytoskeletal structures associated with basal bodies in Paramecium

The presence of phosphorylated proteins associated with microtubule organizing centers in tissue culture cells during mitosis has been demonstrated by the use of monoclonal antibodies raised against mitotic HeLa cells [Vandre et al., Proc. Natl. Acad. Sci. U.S.A. 81:4439-4443, 1984]. We report here that in Paramecium two of the mitosis specific antibodies, MPM-1 and MPM-2, decorate throughout the cell cycle all the microtubule organizing centers (MTOCs) located in the cortex and in the oral apparatus (gullet). Immuno-electron microscopy showed that these antibodies labeled the electron-dense material surrounding basal bodies from which several microtubule networks as well as kinetodesmal fibers originate. During mitosis, these antibodies also stained other cortical cytoskeletal structures, the kinetodesmal fibers (MPM-1 and MPM-2) and the epiplasm (MPM-1). Among the different polypeptides recognized by the antibodies on immunoblots, three major ones of 60, 63, and 116 kDa were found to be common to the cortex (where several thousand ciliary basal bodies are anchored) and the oral apparatus (which comprises several hundred basal bodies around which various arrays of cytoplasmic microtubules are organized). Alkaline phosphatase treatment abolished the immunoreactivity of the polypeptides and the labeling observed by immunofluorescence. These results demonstrate that phosphorylated proteins are associated with all the known active microtubule organizing centers present in the cortex throughout the cell cycle of Paramecium. Furthermore they indicate that in Paramecium phosphorylation of proteins could also be involved in the cell cycle dependent dynamics of cortical cytoskeletal structures other than microtubules.     

The formin mDia regulates GSK3beta through novel PKCs to promote microtubule stabilization but not MTOC reorientation in migrating fibroblasts

In migrating cells, external signals polarize the microtubule (MT) cytoskeleton by stimulating the formation of oriented, stabilized MTs and inducing the reorientation of the MT organizing center (MTOC). Glycogen synthase kinase 3beta (GSK3beta) has been implicated in each of these processes, although whether it regulates both processes in a single system and how its activity is regulated are unclear. We examined these issues in wound-edge, serum-starved NIH 3T3 fibroblasts where MT stabilization and MTOC reorientation are triggered by lysophosphatidic acid (LPA), but are regulated independently by distinct Rho GTPase-signaling pathways. In the absence of other treatments, the GSK3beta inhibitors, LiCl or SB216763, induced the formation of stable MTs, but not MTOC reorientation, in starved fibroblasts. Overexpression of GSK3beta in starved fibroblasts inhibited LPA-induced stable MTs without inhibiting MTOC reorientation. Analysis of factors involved in stable MT formation (Rho, mDia, and EB1) showed that GSK3beta functioned upstream of EB1, but downstream of Rho-mDia. mDia was both necessary and sufficient for inducing stable MTs and for up-regulating GSK3beta phosphorylation on Ser9, an inhibitory site. mDia appears to regulate GSK3beta through novel class PKCs because PKC inhibitors and dominant negative constructs of novel PKC isoforms prevented phosphorylation of GSK3beta Ser9 and stable MT formation. Novel PKCs also interacted with mDia in vivo and in vitro. These results identify a new activity for the formin mDia in regulating GSK3beta through novel PKCs and implicate novel PKCs as new factors in the MT stabilization pathway.     

In vitro microtubule-nucleating activity of spindle pole bodies in fission yeast Schizosaccharomyces pombe: cell cycle-dependent activation in xenopus cell-free extracts

The spindle pole body (SPB) is the equivalent of the centrosome in fission yeast. In vivo it nucleates microtubules (MTs) during mitosis, but, unlike animal centrosomes, does not act as a microtubule organizing center (MTOC) during interphase. We have studied the MT-nucleating activity of SPBs in vitro and have found that SPBs in permeabilized cells retain in vivo characteristics. SPBs in cells permeabilized during mitosis can nucleate MTs, and are recognized by two antibodies: anti-gamma-tubulin and MPM-2 which recognizes phosphoepitopes. SPBs in cells permeabilized during interphase cannot nucleate MTs and are only recognized by anti-gamma-tubulin. Interphase SPBs which cannot nucleate can be converted to a nucleation competent state by incubation in cytostatic factor (CSF)-arrested Xenopus egg extracts. After incubation, they are recognized by MPM-2, and can nucleate MTs. The conversion does not occur in Xenopus interphase extract, but occurs in Xenopus interphase extract driven into mitosis by preincubation with exogenous cyclin B. The conversion is ATP dependent and inhibited by protein kinase inhibitors and alkaline phosphatase. Purified, active, cdc2 kinase/cyclin B complex in itself is not effective for activation of MT nucleation, although some interphase SPBs are now stained with MPM-2. These results suggest that the ability of SPBs in vitro to nucleate MTs after exposure to CSF-arrested extracts is activated through a downstream pathway which is regulated by cdc2 kinase.     

Immunoelectron microscopic localization of phosphoproteins associated with the mitotic spindle.

We examined the immunogold staining of microtubules and microtubule organizing centers using an improved silver-enhancement reagent for small (1-1.4 nm) gold-conjugated secondary antibodies. First, the staining properties of different commercial preparations of gold-labeled antibodies were compared for sample penetration, label uniformity, and labeling density, and Nanogold 1.4-nm gold-conjugated F(ab') was found to be superior to the other probes examined. However, in samples examined for the localization of alpha- and beta-tubulin, gold staining did not extend through the pericentriolar material nor were the centrioles labeled. This apparent lack of centrosomal staining was not due to problems associated with penetration of the antibody probes, since staining adjacent to and within the centriolar cylinder was observed when phosphoprotein antigens recognized by the MPM-2 antibody were localized. The MPM-2 antibodies also localized to mitotic kinetochores, kinetochore fibers, and midbodies, in addition to mitotic centrosomes. The level of MPM-2 staining of the centrosome varied through the cell cycle. At interphase, this staining was restricted within the centriolar cylinder, whereas in mitotic cells extensive staining throughout the pericentriolar material was also observed. These results established the close relationship of MPM-2-reactive phosphoproteins with the centrosome, and suggest that this technique may be useful for ultrastructural localization of other cytoskeletal proteins.     

Cilia regeneration in starved tetrahymena: an inducible system for studying gene expression and organelle biogenesis.

Deciliated starved Tetrahymena recover motility with kinetics similar to those of growing cells and, like growing cells, require RNA and protein synthesis for regeneration. Comparisons of polysome profiles and electrophoretic analyses of newly synthesized proteins indicate, however, that the basal level of protein synthesis in starved cells is markedly lower than that in growing cells. This difference allows demonstration of changes in protein synthesis following deciliation of starved cells which cannot be detected (if they occur at all) in growing cells. Deciliation of starved cells induces a specific and orderly program of protein synthesis. The synthesis of an 80,000 dalton protein (deciliation-induced protein, DIP) begins shortly after deciliation, comprises 15% of the protein synthesized from 20-60 min, and declines around 60 min after deciliation, shortly after most cells have begun to regenerate cilia. The synthesis of a 55,000 dalton protein is also induced during regeneration and has been identified as tubulin using a well characterized antibody made to ciliary tubulin. Tubulin synthesis is undetectable during the first hour after deciliation even though 60-80% of the cells regain mobility and regenerate short but clearly visible cilia. Tubulin synthesis begins 60 min after deciliation and continues for 2 hr. At its peak, tubulin comprises 7-8% of the protein synthesized. The results of actinomycin D addition at different times after deciliation suggest that RNA required for DIP synthesis is synthesized early (0-30 min), while RNA required for tubulin is synthesized later and over a longer period (30-90 min). Thus deciliation of starved cells, an event occurring at the cell periphery, initiates a well defined and reproducible series of events culminating in cilia formation. This system should be useful in elucidating the molecular mechanisms regulating gene expression and organelle biogenesis in Tetrahymena.     

Phosphorylation in vivo of Ribosomes in Tetrahymena pyriformis.

Phosphorylation of ribosomal proteins in vivo was studied in exponentially growing and starved cells of the ciliated protozoan, Tetrahymena pyriformis. No phosphorylation of ribosomal proteins could be demonstrated in cells growing exponentially in complex nutrient media. However, when Tetrahymena cells were transferred into a non-nutrient medium, pronounced phosphorylation of a single ribosomal protein was observed. During two-dimensional polyacrylamide gel electrophoresis the phosphorylated ribosomal protein migrated in a manner virtually identical to that of the phosphorylated ribosomal protein S6 of rat liver. The phosphorylated ribosomal protein has a molecular weight of 38000 as estimated by dodecylsulfate polyacrylamide gel electrophoresis. Thus, the phosphorylated ribosomal protein found in starved Tetrahymena is apparently homologous with the ribosomal protein which is predominantly phosphorylated in higher eukaryotes. When phosphorylated ribosomes were dissociated by treatment with high concentration of KCl, the phosphorylated protein was found only on the small subunit. If dissociation was achieved by dialysis against a buffer low in MgCl2, the phosphorylated protein was distributed almost equally between the two subunits. This indicates that the phosphorylated ribosomal protein is located at the interface between the two subunits.