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Adult human mesenchymal stem cells are primary, multipotent cells capable of differentiating to osteocytic, chondrocytic, and adipocytic lineages when stimulated under appropriate conditions. To characterize the molecular mechanisms that regulate osteogenic differentiation, we examined the contribution of mitogen-activated protein kinase family members, ERK, JNK, and p38. Treatment of these stem cells with osteogenic supplements resulted in a sustained phase of ERK activation from day 7 to day 11 that coincided with differentiation, before decreasing to basal levels. Activation of JNK occurred much later (day 13 to day 17) in the osteogenic differentiation process. This JNK activation was associated with extracellular matrix synthesis and increased calcium deposition, the two hallmarks of bone formation. Inhibition of ERK activation by PD98059, a specific inhibitor of the ERK signaling pathway, blocked the osteogenic differentiation in a dose-dependent manner, as did transfection with a dominant negative form of MAP kinase kinase (MEK-1). Significantly, the blockage of osteogenic differentiation resulted in the adipogenic differentiation of the stem cells and the expression of adipose-specific mRNAs peroxisome proliferator-activated receptor gamma2, aP2, and lipoprotein lipase. These observations provide a potential mechanism involving MAP kinase activation in osteogenic differentiation of adult stem cells and suggest that commitment of hMSCs into osteogenic or adipogenic lineages is governed by activation or inhibition of ERK, respectively.

Analysis of the carboxymethylated subunit of human cartilage oligomeric matrix protein (COMP) by matrix-assisted laser desorption time-of-flight mass spectrometry indicated a protonated molecular mass of 86949 ± 149 Da, compared with 83547.0 Da calculated from the sequence. Treatment withN-glycanase caused a reduction in mass of 3571 ± 219 Da, but there was no loss of mass after treatment withO-glycanase or neuraminidase. Peptides containing two putative sites of N-glycosylation were purified and characterized. Analysis of the masses of these afterN-glycanase treatment indicated that one was substituted at Asn-101 with an oligosaccharide of mass 1847.2 ± 6.6 Da, and the other was unsubstituted at Asn-124. The remaining site of attachment, at Asn-721, was, therefore, also substituted with an oligosaccharide of mass 1724 ± 226 Da. Analysis of the total monosaccharide content by chemical methods indicated that there were no additional oligosaccharide substituents. The MALDI-TOF mass spectra of COMP from bovine fetal and adult cartilage were compared, indicating a more heterogeneous pattern of substitution at Asn-101 in the fetal form. Since COMP is distributed throughout the pericellular and territorial environments in developing cartilage but occupies the interterritorial zone in mature cartilage, these changes in glycosylation may allow for different intermolecular interactions.

Characteristic of chronic myelogenous leukemia (CML) is the presence of the chimeric p210(bcr-abl) protein possessing elevated protein tyrosine kinase activity relative to normal c-abl tyrosine kinase. Hematopoietic progenitors isolated from CML patients in the chronic phase contain a constitutively tyrosine-phosphorylated protein that migrates at 62 kDa by SDS-PAGE and associates with the p120 ras GTPase-activating protein (GAP). We have purified p62(dok) from a hematopoietic cell line expressing p210(bcr-abl). p62(dok) is a novel protein with features of a signaling molecule. Association of p62(dok) with GAP correlates with its tyrosine phosphorylation. p62(dok) is rapidly tyrosine-phosphorylated upon activation of the c-Kit receptor, implicating it as a component of a signal transduction pathway downstream of receptor tyrosine kinases.

The interferon-induced RNA-dependent protein kinase PKR is found in cells in a latent state. In response to the binding of double-stranded RNA, the enzyme becomes activated and autophosphorylated on several serine and threonine residues. Consequently, it has been postulated that autophosphorylation is a prerequisite for activation of the kinase. We report the identification of PKR sites that are autophosphorylated in vitro concomitantly with activation and examine their roles in the activation of PKR. Mutation of one site, threonine 258, results in a kinase that is less efficient in autophosphorylation and in phosphorylating its substrate, the initiation factor eIF2, in vitro. The mutant kinase is also impaired in vivo, displaying reduced ability to inhibit protein synthesis in yeast and mammalian cells and to induce a slow-growth phenotype in Saccharomyces cerevisiae. Mutations at two neighboring sites, serine 242 and threonine 255, exacerbated the effect. Taken together with earlier results (S. B. Lee, S. R. Green, M. B. Mathews, and M. Esteban, Proc. Natl. Acad. Sci. USA 91:10551-10555, 1994), these data suggest that the central part of the PKR molecule, lying between its RNA-binding and catalytic domains, regulates kinase activity via autophosphorylation.

The transforming growth factor beta (TGF-beta) family of growth modulators play critical roles in tissue development and maintenance. Recent data suggest that individual TGF-beta isoforms (TGF-beta 1, -beta 2 and -beta 3) have overlapping yet distinct biological actions and target cell specificities, both in developing and adult tissues. The TGF-beta 3 isoform was purified to homogeneity from both natural and recombinant sources and characterized by laser desorption mass spectrometry, by protein sequencing, by amino acid analysis and by biological activity. TGF-beta 3 was the major TGF-beta isoform in umbilical cord (230 ng/g), and was physically and biologically indistinguishable from recombinant TGF-beta 3 and from the tumor growth inhibitory (TGI) protein found in umbilical cord. Immunohistochemistry using antipeptide TGF-beta 3 specific antibody showed TGF-beta 3 localization in perivascular smooth muscle.

Protein kinase CKII (formerly casein kinase II) can be isolated as a heterotetramer, containing two catalytic (alpha or alpha') and two regulatory (beta) subunits. We have characterized the forms of CKII in HeLa cells using antibodies specific for the alpha or alpha' subunits. Following metabolic labeling with [35S]methionine, whole cell soluble extracts were analyzed by immunoprecipitation and gel electrophoresis. Both alpha and alpha' coprecipitate with beta and with each other. However, when extracts are depleted of alpha, a pool of CKII containing only alpha' and beta is identified. Similarly, depletion of alpha' revealed a pool exclusively of alpha and beta. Therefore, we propose that there are three distinct isoforms of CKII within HeLa cells with different catalytic subunit stoichiometries (alpha 2 beta 2, alpha alpha' beta 2, and alpha' 2 beta 2). With our immunodepletion procedure we have characterized the isoforms by activity analysis, turnover of pulse-labeled subunits, and by localization in subcellular fractions obtained from labeled cells. We have also analyzed complex formation between the catalytic and regulatory subunits by examining the differences in the rate of signal incorporation into subunits in immunoprecipitates obtained from continuously labeled and pulse-labeled cells. We have found that the alpha 2 beta 2 and alpha alpha' beta 2 isoforms assemble relatively slowly (12-16 h), whereas complex formation of the alpha' 2 beta 2 isoform occurs more rapidly (2-4 h). Analysis of isoform complex formation in subcellular fractions from pulse-labeled cells revealed that the majority of nuclear CKII is assembled in the nucleus from free catalytic and regulatory subunit polypeptides.

Mitogen-activated protein kinase kinase 1 (MKK1), a dual-specificity tyrosine/threonine protein kinase, has been shown to be phosphorylated and activated by the raf oncogene product as part of the mitogen-activated protein kinase cascade. Here we report the phosphorylation and inactivation of MKK1 by phosphorylation on threonine 286 and threonine 292. MKK1 contains a consensus phosphorylation site for p34cdc2, a serine/threonine protein kinase that regulates the cell division cycle, at Thr-286 and a related site at Thr-292. p34cdc2 catalyzes the in vitro phosphorylation of MKK1 on both of these threonine residues and inactivates MKK1 enzymatic activity. Both sites are phosphorylated in vivo as well. The data presented in this report provide evidence that MKK1 is negatively regulated by threonine phosphorylation.

We report a method for optimizing the specificity of product formation in the polymerase chain reaction (PCR). This technique is based on the use of dimethyl sulfoxide (DMSO) and takes into account primer Tm. The reduction in Tm by DMSO is directly correlated with renaturation temperature such that a DMSO gradient reflects a temperature gradient. We use this relationship to show that optimum product formation usually occurs at or within several degrees of the midpoint Tm of a given primer pair. We illustrate these correlations using three examples deriving PCR products from a human cDNA library, representing the casein kinase II alpha and beta subunits as well as the 5' untranslated region for the beta subunit. By following product formation as a function of renaturation temperature, we postulate rules for cycle design based on primer Tm. Implications for the use of degenerate primers are discussed.

The activity of p34cdc2 kinase is regulated in the phases of vertebrate cell cycle by mechanisms of phosphorylation and dephosphorylation. In this paper, we demonstrate that casein kinase II (CKII) phosphorylates p34cdc2 in vivo and in vitro at Ser39 during the G1 phase of HeLa cell division cycle. Human p34cdc2 shows a typical phosphorylation sequence motif site for CKII at Ser39 (ES39EEE). In our experiments, either p34cdc2 expressed and purified from bacteria or p34cdc2 immunoprecipitated from HeLa cells enriched in G1 by elutriation were substrates for in vitro phosphorylation by CKII. Phosphoamino acid analysis, N-chlorosuccinimide mapping, and two-dimensional tryptic mapping of p34cdc2 phosphorylated in vitro were performed to determine the phosphorylation site. A synthetic peptide spanning residues 33-50 of human p34cdc2, including the CKII site, was used to map the site. In addition, phosphorylation at Ser39 also occurs in vivo, since p34cdc2 is phosphorylated during G1 on serine, and its two-dimensional tryptic map shows two phosphopeptides that comigrate exactly with the synthetic peptides used as standard.

c-jun is a member of the family of immediate-early genes whose expression is induced by factors such as serum stimulation, phorbol ester, and differentiation signals. Here we show that increased Jun synthesis after serum stimulation is accompanied by a concomitant increase in phosphorylation. Several serine-threonine kinases were evaluated for their ability to phosphorylate Jun in vitro. p34cdc2, protein kinase C, casein kinase II, and pp44mapk phosphorylated Jun efficiently, whereas cyclic AMP-dependent protein kinase and glycogen synthase kinase III did not. The sites phosphorylated by p34cdc2 were similar to those phosphorylated in vivo after serum induction. The major sites of phosphorylation were mapped to serines 63, 73, and 246. Phosphorylation of full-length Jun with several kinases did not affect the DNA-binding activity of Jun homodimers or Fos-Jun heterodimers. Comparison of the DNA binding and in vitro transcription properties of wild-type and mutated proteins containing either alanine or aspartic acid residues in place of Ser-63, -73, and -246 revealed only minor differences among homodimeric complexes and no differences among Fos-Jun heterodimers. Thus, phosphorylation of Jun did not produce a significant change in dimerization, DNA-binding, or in vitro transcription activity. The regulatory role of phosphorylation in the modulation of Jun function is likely to be considerably more complex than previously suggested.

Several small, acid-soluble, basic peptides with anti-microbial properties have been isolated from maize (inbred B73) kernels. One of these peptides (MBP-1) has been purified to homogeneity and characterized. The peptide has a molecular weight of 4127.08 as determined by plasma desorption mass spectroscopy, has no free cysteines, and is predominantly alpha-helical as determined by circular dichroism. The primary sequence of the peptide (33 residues) has been determined by Edman degradation and shows no homology to the thionins, a group of cysteine-rich peptides found in some cereals including wheat, barley, and sorghum, as well as several dicot species. Like the thionins, however, MBP-1 has been found to have antimicrobial properties in vitro. MBP-1 inhibits spore germination or hyphal elongation of several plant pathogenic fungi, including two seed pathogens of maize (Fusarium moniliforme Sheld. and Fusarium graminearum (Gibberella zeae (Schw.) Petsch)), and several bacteria, including a bacterial pathogen of maize (Clavibacter michiganense ssp. nebraskense). A synthetic MBP-1 peptide, air-oxidized and purified by reverse phase chromatography, was equally antifungal as compared with the naturally occurring peptide.

Heparin-binding growth-associated molecule (HB-GAM) was purified from adult bovine brain and chicken heart. The yield of HB-GAM is increased by 5- to 10-fold when 250 mM NaCl is added to the homogenization buffer, indicating that HB-GAM may exist as a complex with an insoluble component of the tissue. The complete amino acid sequence of the brain-derived HB-GAM was established by automated Edman degradation of the intact protein and chemically or enzymatically derived fragments. The mass of bovine HB-GAM as determined by plasma desorption time-of-flight mass spectrometry is 15,291 mass units, which compares favorably with the calculated mass of 15,289 based on the amino acid sequence. Therefore, HB-GAM has not undergone any major post-translational modifications other than cleavage of the signal peptide. These results indicate that previous amino acid sequence analysis of this protein was carried out using truncated HB-GAM. Full-length HB-GAM is not a mitogen for Balb/3T3 clone A31, Balb MK, NRK, or human umbilical vein endothelial cells. HB-GAM does, however, have adhesive properties and neurite extension activity for chick embryo cerebral cortical derived neurons when presented to these cells as a substrate. HB-GAM had little neurite extension activity when presented as a soluble factor.

Synthetic peptide substrates for the cell division cycle regulated protein kinase, p34cdc2, have been developed and characterized. These peptides are based on the sequences of two known substrates of the enzyme, Simian Virus 40 Large T antigen and the human cellular recessive oncogene product, p53. The peptide sequences are H-A-D-A-Q-H-A-T-P-P-K-K-K-R-K-V-E-D-P-K-D-F-OH (T antigen) and H-K-R-A-L-P-N-N-T-S-S-S-P-Q-P-K-K-K-P-L-D-G-E-Y-NH2 (p53), and they have been employed in a rapid assay of phosphorylation in vitro. Both peptides show linear kinetics and an apparent Km of 74 and 120 microM, respectively, for the purified human enzyme. The T antigen peptide is specifically phosphorylated by p34cdc2 and not by seven other protein serine/threonine kinases, chosen because they represent major classes of such enzymes. The peptides have been used in whole cell lysates to detect protein kinase activity, and the cell cycle variation of this activity is comparable to that measured with specific immune and affinity complexes of p34cdc2. In addition, the peptide phosphorylation detected in mitotic cells is depleted by affinity adsorption of p34cdc2 using either antibodies to p34cdc2 or by immobilized p13, a p34cdc2-binding protein. Purification of peptide kinase activity from mitotic HeLa cells yields an enzyme indistinguishable from p34cdc2. These peptides should be useful in the investigation of p34cdc2 protein kinase and their regulation throughout the cell division cycle.

We have recently reported that protein kinase CK2 phosphortylates both in vivo and in vitro residue serine-46 of the cell cycle regulating protein Cdc28 of budding yeast Saccharomyces cerevisiae, confirming a previous observation that the same site is phosphorylated in Cdc2/Cdk1, the human homolog of Cdc28. In addition, S. cerevisiae in which serine-46 of Cdc28 has been mutated to alanine show a decrease of 33% in both cell volume and protein content, providing the genetic evidence that CK2 is involved in the regulation of budding yeast cell division cycle, and suggesting that this regulation may be brought about in G1 phase of the mammalian cell cycle. Here, we extended this observation reporting that the mutation of serine-46 of Cdc28 to glutamic acid doubles, at least in vitro, the H1-kinase activity of the Cdc28/cyclin A complex. Since this mutation has only little effects on the cell size of the cells, we hypothesize multiple roles of yeast CK2 in regulating the G1 transition in budding yeast.

To investigate the biological function of CKII, we have identified proteins that interact with the subunits of CKII using the yeast two-hybrid system. Here we report that SAG, an antioxidant protein containing Ring-H2 finger motif, is a cellular partner associating with the beta subunit of CKII. SAG does not interact with the alpha subunit of CKII. Analysis of SAG deletion mutants indicates that the Ring-H2 motif of SAG is necessary and sufficient for its binding to the beta subunit of CKII. Recombinant SAG can be phosphorylated by CKII in vitro, providing evidence that the beta subunit mediates the interaction of CKII enzyme with substrate proteins. Overlay experiment shows that SAG and the beta subunit of CKII associate directly in vitro and that CKII-mediated phosphorylation of SAG does not affect the interaction between SAG and the beta subunit of CKII. Northern blot analysis indicates that both SAG and the beta subunit of CKII were relatively rich in human heart, liver, skeletal muscle, and pancreas, but were detected in only trace amounts in brain, placenta, and lung. Our present results suggest that CKII may play a role in the regulation of SAG function.

Human mesenchymal stem cells are thought to be multipotent cells, which are present in adult marrow, that can replicate as undifferentiated cells and that have the potential to differentiate to lineages of mesenchymal tissues, including bone, cartilage, fat, tendon, muscle, and marrow stroma. Cells that have the characteristics of human mesenchymal stem cells were isolated from marrow aspirates of volunteer donors. These cells displayed a stable phenotype and remained as a monolayer in vitro. These adult stem cells could be induced to differentiate exclusively into the adipocytic, chondrocytic, or osteocytic lineages. Individual stem cells were identified that, when expanded to colonies, retained their multilineage potential.

Protein kinase CKII (CKII) is a heterotetramer composed of two catalytic (alpha or alpha') and two regulatory (beta) subunits. Using the yeast two-hybrid system, we have identified the highly basic, ribosomal protein L41 as a cellular protein capable of interacting with the beta subunit of CKII. We show, furthermore, using purified proteins, that L41 protein and CKIIbeta associate directly in vitro. L41 protein is not a substrate for CKII phosphorylation, and it does not stimulate CKII activity with either beta-casein or synthetic peptide substrate (RRREEETEEE). However, L41 protein stimulates the phosphorylation of DNA topoisomerase IIalpha by CKII by 2.5 times. Additionally, L41 protein enhances the autophosphorylation of CKIIalpha. The data indicate that L41 protein associates with CKII and can modulate its activity toward a specific substrate or substrates. The direct interaction of CKIIbeta with ribosomal proteins also suggests that CKIIbeta itself or CKII holoenzyme may be involved in ribosome assembly or translational control.

Casein kinase II (CKII) usually exists as a heterotetramer with alpha 2 beta 2, alpha alpha'beta 2, or alpha'2 beta 2. The alpha or alpha' subunits catalyze protein phosphorylation, whereas the function of the beta subunit remains unclear. One of the possible functions of the beta subunit may be to mediate the interaction of the catalytic subunit with target proteins. To identify proteins capable of associating with the beta subunit in vivo, we have used a two-hybrid system. One protein identified is human ribosomal protein L5. The protein L5 does not interact with the alpha or alpha' subunits of CKII, supporting the idea that the beta subunit can determine a substrate specificity of CKII. These results furthermore suggest a novel role for CKII in ribosomal L5 phosphorylation, in ribosomal assembly, or ribosomal transport in the intact cells. The protein L5 may act as a regulator of the activity or subcellular localization of CKII.

We are developing methods to identify Gi-phase substrates of cyclin-Cdk: complexes. In earlier years of this project, we optimized two-dimensional gels for visualization of phosphorylation of Cdc28 substrates, and we developed monoclonal antibodies with reactivity to phospho-Ser-Pro or phospho-Thr- Pro. We identified one important in vivo substrate, Sic 1. However, it appears that these methods by themselves are insufficiently powerful to identify many substrates, since most substrates seem to be very rare. We have therefore supplemented these methods with three other approaches: (1) purification of affinity tagged cyclins, to look for co-purifying proteins; (2) complete genome two-hybrid screens with Cdc28 and with Gi cyclins, to look for interacting proteins; and (3) genome-wide analysis of cell cycle regulated transcription, on the basis that some important substrates might be regulated at the transcriptional level as well as at the activity level. All three approaches are going very well, and we have excellent candidates for new substrates, which will be characterized in part using the 2D gel system.

Epithelial- and haematopoietic-cell growth-inhibitory activities have been identified in the conditioned medium of the human peripheral neuroepithelioma cell line A673. An A673-cell-derived growth-inhibitory activity was previously fractionated into two distinct components which inhibited the proliferation of human carcinoma and leukaemia cells in culture. One inhibitory activity was shown to comprise interleukin-1 alpha (IL-1 alpha). Here, we have purified to homogeneity a distinct activity which inhibited the growth of the epithelial cells in vitro. Using a combination of protein-sequence analysis and mass spectrometry, we demonstrated that biological activity can be assigned to a dimeric protein with a molecular mass of 25,576 (+/- 4) Da and an N-terminal sequence identical with that of transforming growth factor-beta 1 (TGF-beta 1). Further characterization of the growth inhibitor with TGF-beta-isoform-specific antibodies showed that > 90% of the bioactivity consists of TGF-beta 1 and not TGF-beta 2 or TGF-beta 3. Although A673 cells were growth-inhibited by exogenous TGF-beta 1, we showed that TGF-beta 1 in A673-cell-conditioned media was present in the latent, biologically inactive, form which did not act as an autocrine growth modulator of A673 cells in vitro.

ATP citrate-lyase (CL), acetyl-CoA carboxylase (ACC) and glycogen synthase kinase-3 beta (GSK-3 beta) levels were measured in cytosol from 3T3-L1 cells during differentiation from fibroblasts into fat-cells. Protein levels were estimated from immunoblots using specific antisera. Cytosol from confluent cells contain significant amounts of GSK-3 beta, which fell during differentiation of these cells into adipocytes. CL from confluent cells was found to be mostly in the form of a single protein band of apparent mass 110 kDa. Levels of CL and ACC increased during cell differentiation into adipocytes. During the first 3 days of differentiation, CL migration changed, and it was expressed as a complex of protein bands of apparent mass 110 kDa, 113 kDa and 115 kDa. At later stages of differentiation, when these cells had assumed the phenotype of fat-cells, they expressed CL mainly as protein bands of 110 and 113 kDa. When samples containing these bands were treated with alkaline phosphatase, the 113 kDa protein band collapsed into the 110 kDa species. This suggests that the slower-migrating species of CL is a higher-order phosphorylation state of the same protein. Furthermore, when purified CL, mostly expressed as the 110 kDa species, was phosphorylated with cyclic AMP-dependent protein kinase alone or together with GSK-3 and resolved by SDS/PAGE, the phosphorylated CL now migrated more slowly as the 113 kDa and 115 kDa forms. CL phosphorylation was hormone-regulated, since, in samples from fat-cells that had the complex two-band pattern, when cultured in medium without serum or hormones, CL migration reverted to a single band of 110 kDa, similar to confluent cells. Treatment of these ‘down-regulated’ cells with insulin rapidly induced substantial amounts of the 113 kDa species, with a concomitant decrease in the 110 kDa species.

Protein F1/GAP43 is neuron-specific, associated with neurite outgrowth during development and a substrate for PKC. This protein is present in high levels in serotonergic neurons which in culture sprout in response to the glial-derived S100b, the beta-beta homodimer. As an initial step in determining whether S100b acts on F1/GAP43 we studied the regulation by S100b of PKC phosphorylation of F1/GAP43. Either the S100b or a mixture of S100a and S100b, both from a brain glial cell source, inhibited in vitro phosphorylation of purified F1/GAP43 by purified PKC in a dose-dependent manner. Using recombinant PKC subtypes, purified S100b preferentially inhibited the F1/GAP43 phosphorylation by the beta subtype. The IC50 of S100b for beta I and beta II PKC was 8 microM while for alpha and gamma PKC it was 64 microM. S100b inhibition was thus subtype-selective. Histone III-S phosphorylation by the four PKC subtypes was not inhibited by S100b. S100b inhibition was thus substrate-selective. Moreover, the effect of S100b on phosphorylation could not be explained by a direct inhibition of kinase activity. Together with earlier studies implicating a role for S100 in synaptic plasticity and neurite outgrowth, the present results suggest that S100b may regulate such functions through its inhibition of neuron-specific PKC substrate (F1/GAP43) phosphorylation. The regulation of this neuron-specific substrate phosphorylation by glial S100 suggests the potential for a novel neuro-glial interaction. Finally, the location of S100 gene on chromosome 21, trisomic in Down's syndrome, and over-expressed in this disorder, as well as in Alzheimer's disease, suggests a link to cognitive impairments in human.

We have developed specific antibodies to synthetic peptide antigens that react with the individual subunits of casein kinase II (CKII). Using these antibodies, we studied the localization of CKII in asynchronous HeLa cells by immunofluorescence and immunoelectron microscopy. Further studies were done on HeLa cells arrested at the G1/S transition by hydroxyurea treatment. Our results indicate that the CKII alpha and beta subunits are localized in the cytoplasm during interphase and are distributed throughout the cell during mitosis. Further electron microscopic investigation revealed that CKII alpha subunit is associated with spindle fibers during metaphase and anaphase. In contrast, the CKII alpha' subunit is localized in the nucleus during G1 and in the cytoplasm during S. Taken together, our results suggest that CKII may play significant roles in cell division control by shifting its localization between the cytoplasm and nucleus.

The regulation of DNA replication during the eukaryotic cell cycle was studied in a system where cell free replication of simian virus 40 (SV40) DNA was used as a model for chromosome replication. A factor, RF-S, was partially purified from human S phase cells based on its ability to activate DNA replication in extracts from G1 cells. RF-S contained a human homologue of the Schizosaccharomyces pombe p34cdc2 kinase, and this kinase was necessary for RF-S activity. The limiting step in activation of the p34 kinase at the G1 to S transition may be its association with a cyclin since addition of cyclin A to a G1 extract was sufficient to start DNA replication. These observations suggest that the role of p34cdc2 in controlling the start of DNA synthesis has been conserved in evolution.

Neurons express high levels of a variant form of the c-src gene product, denoted pp60c-src(+), which contains a 6 amino acid insert in the amino-terminal half of the c-src protein. We have determined the localization of pp60c-src(+) in neurons using an affinity-purified anti- peptide antibody, referred to as affi-SB12, that exclusively recognizes this neuron-specific form of the c-src gene product. Using affi-SB12, we examined the distribution of pp60c-src(+) by immunoperoxidase staining of sections through adult rat brains, pp60c-src(+) was widely distributed in rat brain and appeared to be differentially expressed in subpopulations of neurons. The majority of immunoreactive neurons was found in the mesencephalon, cerebellum, pons, and medulla. Telencephalic structures that contained substantial populations of pp60c-src(+)-immunoreactive neurons included layer V of the cerebral cortex and the ventral pallidum. Within individual neurons, pp60c- src(+) immunoreactivity was localized to the cell soma and dendritic processes, while labeling of axons and nerve terminals (puncta) was not as readily detected. Dense accumulations of immunoreactive axons were rare, being most prominent in portions of the inferior and superior olive, and in the spinal trigeminal nucleus. While the regional distribution of pp60c-src(+) immunoreactivity does not correlate with any specific neuronal cell type or first messenger system, this unique pattern of expression of pp60c-src(+) suggests the existence of a previously uncharacterized functional organization within the brain. Furthermore, the localization of this neuron-specific tyrosine kinase in functionally important areas of the nerve cell, namely, dendritic processes, axons, and nerve terminals, suggests that pp60c-src(+) may regulate pleiotropic functions in specific classes of neurons in the adult central nervous system.

S100 beta is a multifunctional protein that is found in large amounts in astrocytes and a number of other tissues. In the developing nervous system, S100 beta is secreted from proliferating astrocytes during the time of neurite outgrowth from cortical neurons. The secreted form has neurotrophic activity on primary neurons and neuroblastoma cells. The neurotrophic activity is sensitive to reduction of disulfide bonds, and appears to be a disulfide dimer of S100 beta. The accumulation of S100 beta in mature glial cells is associated with microtubule network. The transfection and expression of cDNA for S100 beta in mammalian cells confers neurotrophic activity on extracts of these cells. Based on our observations of a neurotrophic activity for S100 beta, the occurrence of the gene for S100 beta on human chromosome 21 (Allore et al., 1988) and the elevated levels of S100 beta-containing cells in AD and DS brains (Griffin et al., 1989), we suggest that S100 beta plays a role in the abnormal development of the nervous system in DS and the degeneration of central neurons in AD. It is essential at the present stage to demonstrate the action of S100 beta in vivo, and to construct animal models, such as transgenic mice, that overexpress S100 beta. Such models will allow the dissection of the role of S100 beta in the developing and degenerating central nervous system.

We have tested the effects of serum-stimulated growth of quiescent WI38 human lung fibroblasts on cellular casein kinase II (CK-II) activity. Using the casein kinase II synthetic substrate RRREEETEEE we find a transient 6-fold elevation in CK-II activity in cell homogenates within 30 min following serum stimulation. Additional cycles of CK-II activation and inactivation are seen at 12 and 24 h after stimulation. The oscillations in CK-II activity are largely independent of de novo protein synthesis, and, thus, are likely to reflect cycles of post-translational activation and inhibition of the cellular kinase pool. In contrast to the activity profile of CK-II, we find that cyclic AMP-dependent protein kinase is rapidly inhibited upon serum-stimulation of WI38 cells. These results demonstrate that CK-II activity is subject to unique cellular regulation during proliferation and are consistent with the postulate that CK-II plays an important role in cell growth.

An insulin-like growth factor II variant (IGF-II variant) was purified from Cohn fraction IV1 of human plasma by ion exchange, gel filtration, and reversed-phase high pressure liquid chromatography. The amino-terminal sequence of the first 35 amino acid residues showed a replacement of Ser-29 of IGF-II with the tetrapeptide Arg-Leu-Pro-Gly of IGF-II variant. Peptides isolated and sequenced after digestion with endoproteinase Asp-N and endoproteinase Glu-C disclosed no differences with the sequence predicted from an IGF-II variant cDNA clone isolated by Jansen, M., van Shaik, F. M. A., van Tol, H., Van den Brande, J. L., and Sussenbach, J. S. (1985) FEBS Lett., 179, 243-246. The molecular ion of intact IGF-II variant was 7809.4 mass units, as measured by plasma desorption mass spectrometry. This is in close agreement with the molecular ion of 7812.8 mass units calculated from the determined sequence and indicates the entire amino acid sequence had been accounted for. Binding of IGF-II variant to purified insulin-like growth factor I (IGF-I) receptors demonstrated a 2-3-fold lower affinity for this receptor compared with IGF-I or IGF-II. The dissociation constants for IGF-I, IGF-II, and IGF-II variant are 0.23, 0.38, and 0.80 nM, respectively. In a growth assay, the concentration of IGF-II and IGF-II variant required to stimulate the half-maximal growth of MCF-7 cells was 4 and 13 nM, respectively. Finally, the amount of IGF-II variant that can be purified by this method constitutes approximately 25% of the total IGF-II isolated from Cohn fraction IV1 of human plasma.

We have previously described a simple two-step purification technique to isolate alpha 2-adrenergic receptors from the rat adrenocortical carcinoma (Jaiswal, R. K. and Sharma, R. K. (1985) Biochem. Biophys. Res. Commun. 130, 58-64). Utilizing this technique we have now achieved approximately 77,000-fold purification to apparent homogeneity of alpha 2-adrenergic receptors from human platelets. We have compared the biochemical characteristics of these receptors with those from the rat, which were purified approximately 40,000-fold to homogeneity. The [125I] receptor proteins from two sources showed: (a) a single radioactive band with a Mr of 64,000 as evidenced by one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE); and (b) a single symmetrical peak with a pI of 4.2 by isoelectric focusing polyacrylamide gel electrophoresis. Both proteins showed typical alpha 2-adrenergic binding characteristics with specific binding activities of 13.85 nmol/mg and 14.17 nmol/mg protein. These values are close to the theoretical binding activity of 15.6 nmol/mg protein for 1 mol of the ligand binding 1 mol of the receptor protein. These results attest to the purity of the receptors, to its Mr of 64,000, and to its acidic nature. However, the peptide maps of the radioiodinated alpha 2-adrenergic receptors from rat adrenocortical carcinoma and human blood platelets reveal some distinct differences which may relate to the differences in the pharmacological specificities between rodent and non-rodent alpha 2-adrenergic receptors.