Cytoskeletal responsiveness to progestins is dependent on progesterone receptor A levels.

Changes in the cell cytoskeleton occur in cell transformation and recent data suggest the involvement of ovarian hormones, which are implicated in cancer development and progression. In human breast and endometrial tumors, there is disrupted expression of progesterone receptor (PR) isoforms and predominance of one isoform, usually PRA. PRA predominance is an early event in carcinogenesis, and in cancers is associated with poor clinical features. Overexpression of PRA in vitro causes altered progestin regulation of cell morphology, suggesting that PRA overexpression may provoke deleterious changes in cell functioning. This study aimed to identify pathways of cytoskeleton regulation responsive to progestins and to determine whether these are perturbed when PRA is overexpressed to the levels seen in cancers. Progestin treatment of PR-positive breast cancer cells caused increased cell surface area whereas after induction of a stably integrated PRA construct, cells became rounded and the cell surface was decreased. The effect of PRA induction on cell rounding was reversed by the anti-progestin RU38486. Altered tropomyosin (Tm) isoforms were implicated in these morphological differences, as there was a PRA-mediated alteration in Tm5 isoform levels, and transfection of Tm5a mimicked progestin-mediated cell rounding in PRA-overexpressing cells. Ezrin was redistributed from the membrane to cytoplasmic locations in the presence of progestin, and discrete focal localization was evident in cells with PRA predominance. Progestin effects on the cytoskeleton in PRA-overexpressing cells provide evidence for novel endocrine regulation of aspects of actin microfilament composition, suggesting that changes in the cytoskeleton known to be associated with cancer development and progression may be regulated in part by altered PRA expression which develops early in carcinogenesis.

Functional analysis of the actin-binding protein, tropomyosin 1, in neuroblastoma.

Tropomyosin 1 (TM1) is downregulated in a number of transformed cell types, and exogenous expression of TM1 can restore actin organisation and reverse cellular transformation. We find that TM1 is also downregulated in human neuroblastoma cell lines, correlating with increasing malignancy. However, exogenous TM1 does not restore actin cytoskeleton organisation in neuroblastoma cells.

Adeno-associated virus and lentivirus vectors mediate efficient and sustained transduction of cultured mouse and human dorsal root ganglia sensory neurons.

Peripheral nervous system (PNS) sensory neurons are directly involved in the pathophysiology of numerous inherited and acquired neurological conditions. Therefore, efficient and stable gene delivery to these postmitotic cells has significant therapeutic potential. Among contemporary vector systems capable of neuronal transduction, only those based on herpes simplex virus have been extensively evaluated in PNS neurons. We therefore investigated the transduction performance of recombinant adeno-associated virus type 2 (AAV) and VSV-G-pseudotyped lentivirus vectors derived from human immunodeficiency virus (HIV-1) in newborn mouse and fetal human dorsal root ganglia (DRG) sensory neurons. In dissociated mouse DRG cultures both vectors achieved efficient transduction of sensory neurons at low multiplicities of infection (MOIs) and sustained transgene expression within a 28-day culture period. Interestingly, the lentivirus vector selectively transduced neurons in murine cultures, in contrast to human cultures, in which Schwann and fibroblast-like cells were also transduced. Recombinant AAV transduced all three cell types in both mouse and human cultures. After direct microinjection of murine DRG explants, maximal transduction efficiencies of 20 and 200 transducing units per neuronal transductant were achieved with AAV and lentivirus vectors, respectively. Most importantly, both vectors achieved efficient and sustained transduction of human sensory neurons in dissociated cultures, thereby directly demonstrating the exciting potential of these vectors for gene therapy applications in the PNS.

Sorting of tropomyosin isoforms in synchronised NIH 3T3 fibroblasts: evidence for distinct microfilament populations.

The nonmuscle actin cytoskeleton consists of multiple networks of actin microfilaments. Many of these filament systems are bound by the actin-binding protein tropomyosin (Tm). We investigated whether Tm isoforms could be cell cycle regulated during G0 and G1 phases of the cell cycle in synchronised NIH 3T3 fibroblasts. Using Tm isoform-specific antibodies, we investigated protein expression levels of specific Tms in G0 and G1 phases and whether co-expressed isoforms could be sorted into different compartments. Protein levels of Tms 1, 2, 5a, 6, from the alpha Tm(fast) and beta-Tm genes increased approximately 2-fold during mid-late G1. Tm 3 levels did not change appreciably during G1 progression. In contrast, Tm 5NM gene isoform levels (Tm 5NM-1-11) increased 2-fold at 5 h into G1 and this increase was maintained for the following 3 h. However, Tm 5NM-1 and -2 levels decreased by a factor of three during this time. Comparison of the staining of the antibodies CG3 (detects all Tm 5NM gene products), WS5/9d (detects only two Tms from the Tm 5NM gene, Tm 5NM-1 and -2) and alpha(f)9d (detects specific Tms from the alpha Tm(fast) and beta-Tm genes) antibodies revealed 3 spatially distinct microfilament systems. Tm isoforms detected by alpha(f)9d were dramatically sorted from isoforms from the Tm 5NM gene detected by CG3. Tm 5NM-1 and Tm 5NM-2 were not incorporated into stress fibres, unlike other Tm 5NM isoforms, and marked a discrete, punctate, and highly polarised compartment in NIH 3T3 fibroblasts. All microfilament systems, excluding that detected by the WS5/9d antibody, were observed to coalign into parallel stress fibres at 8 h into G1. However, Tms detected by the CG3 and alpha(f)9d antibodies were incorporated into filaments at different times indicating distinct temporal control mechanisms. Microfilaments in NIH 3T3 cells containing Tm 5NM isoforms were more resistant to cytochalasin D-mediated actin depolymerisation than filaments containing isoforms from the alpha Tm(fast) and beta-Tm genes. This suggests that Tm 5NM isoforms may be in different microfilaments to alpha Tm(fast) and beta-Tm isoforms even when present in the same stress fibre. Staining of primary mouse fibroblasts showed identical Tm sorting patterns to those seen in cultured NIH 3T3 cells. Furthermore, we demonstrate that sorting of Tms is not restricted to cultured cells and can be observed in human columnar epithelial cells in vivo. We conclude that the expression and localisation of Tm isoforms are differentially regulated in G0 and G1 phase of the cell cycle. Tms mark multiple microfilament compartments with restricted tropomyosin composition. The creation of distinct microfilament compartments by differential sorting of Tm isoforms is observable in primary fibroblasts, cultured 3T3 cells and epithelial cells in vivo.

Tropomyosin isoform diversity and neuronal morphogenesis.

Tropomyosins (Tm) are a large family of isoforms obtained from multiple genes and by extensive alternative splicing. They bind in the alpha-helical groove of the actin filament and are therefore core components of this extensive cytoskeletal system. In non-muscle cells the Tm isoforms have been implicated in a diversity of processes including cytokinesis, vesicle transport, motility, morphogenesis and cell transformation. Using immunohistochemical localization in cultured primary cortical neurons with an antibody that potentially identifies all non-muscle TM5 gene isoforms compared with one that specifically identifies a subset of isoforms, the possibility was raised that there were considerably more isoforms derived from this gene than the four previously described. Using polymerase chain reaction (PCR) analysis we have now shown that the rat brain generates at least 10 mRNA isoforms using multiple combinations of terminal exons and two internal exons. There is extensive developmental regulation of these isoforms in the brain and there appears to be a switch in the preferential use of the two internal exons 6a to 6b from the embryonic to the adult isoforms. Specific isoforms using alternate carboxyl-terminal exons are differentially localized within the adult rat cerebellum. It is suggested that the tightly regulated spatial and temporal expression of Tm isoforms plays an important role in the development and maintenance of specific neuronal compartments. This may be achieved by isoforms providing unique structural properties to actin-based filaments within functionally distinct neuronal domains.

Structural compartments within neurons: developmentally regulated organization of microfilament isoform mRNA and protein.

The microfilament system is thought to be a crucial cytoskeletal component regulating development and mature function of neurons. The intracellular distribution of the microfilament isoform components, actin and tropomyosin (Tm), in neurons primarily in vivo, has been investigated at both the mRNA and the protein level using isoform specific riboprobes and antibodies. Our in vivo and in vitro studies have identified at least six neuronal compartments based on microfilament isoform mRNA localization: the developing soma, the mature soma, growth cone, developing axon hillock/proximal axon, mature somatodendritic and mature axonal pole soma. Protein localization patterns revealed that the isoforms were frequently distributed over a wider area than their respective mRNAs, suggesting that isoform specific patterns of mRNA targeting may influence, but do not absolutely determine, microfilament isoform location. Tm4 and Tm5 showed identical mRNA targeting in the developing neuron but distinct protein localization patterns. We suggest that in this instance mRNA location may best be viewed as a regulated site of synthesis and assembly, rather than a regulator of protein localization per se. In addition, Tm5 and beta-actin mRNA and protein locations were developmentally regulated, suggesting the possibility that environmental signals modulate targeting of specific mRNAs and their proteins. Thus, developmentally regulated mRNA localization and positional translation may act in concert with protein transport to regulate neuronal microfilament composition and consequently neuronal structure.

Distinct localizations of tropomyosin isoforms in LLC-PK1 epithelial cells suggests specialized function at cell-cell adhesions.

At least eight nonmuscle, nonbrain tropomyosin isoforms have been described. We used antibodies, microinjection, and transfection to characterize their expression and localization in LLC-PK1 kidney epithelial cells and compared them with other cells. Similar to primary enterocytes, LLC-PK1 cells exhibited predominantly TM-1 and TM-3 of the high-molecular-weight (HMW) isoforms; TM-5 and TM-5b of the low-molecular-weight (LMW) isoforms. Neither TM-4 nor TM-5a was detectable in the LLC-PKI cells. Immunofluorescence studies revealed that HMW isoforms were localized only on stress fibers, not adhesion belts, whereas the adhesion belts were stained by LMW isoform antibodies. When exogenous proteins are introduced either by transfection or microinjection, the HMW isoforms do not incorporate into the adhesion belt, whereas the LMW isoforms can incorporate into the stress fibers, thus indicating there are different mechanisms at work for the selective localization. Temporal changes in the microfilament system of the LLC-PK1 cells were studied during differentiation in culture as defined by spectrin expression and F-actin architecture. Western blot analysis indicated that TM-5b is only expressed in the LLC-PK1 cells after a certain degree of maturation in culture, which suggests isoform switching after the cell-cell contacts are developed. Collectively these results demonstrate that epithelial cells express a complex pattern of TM isoforms, which exhibit differential localizations within the cells and different patterns of expression depending on their origin and stage of differentiation. The implication of differential localization of TM isoforms on their specific functions is discussed.

Splicing of two internal and four carboxyl-terminal alternative exons in nonmuscle tropomyosin 5 pre-mRNA is independently regulated during development.

Four nonmuscle tropomyosin isoforms have been reported to be produced from the rat Tm5 gene by alternative splicing (Beisel, K. W., and Kennedy, J. E. (1994) Gene (Amst.) 145, 251-256). In order to detect additional isoforms that might be expressed from that gene, we used reverse transcriptase-polymerase chain reaction assays and evaluated the presence of all product combinations of two alternative internal exons (6a and 6b) and four carboxyl-terminal exons (9a, 9b, 9c, and 9d) in developing and adult rat brain. We identified five different combinations for exon 9 (9a + 9b, 9a + 9c, 9a + 9d, 9c, and 9d), and the exon combinations 9a + 9c and 9a + 9d were previously unreported. Each of these combinations existed with both exon 6a and exon 6b. Thus, the rat brain generates at least 10 different isoforms from the Tm5 gene. Northern blot hybridization with alternative exon-specific probes revealed that these isoforms were also expressed in a number of different adult rat tissues, although some exons are preferentially expressed in particular tissues. Studies of regulation of the 10 different Tm5 isoform mRNAs during rat brain development indicated that no two isoforms are coordinately accumulated. Furthermore, there is a developmental switch in the use of exon 6a to exon 6b from embryonic to adult isoforms. TM5 protein isoforms show a differential localization in the adult cerebellum.

Identification of a dynamic intracellular reservoir of CD86 protein in peripheral blood monocytes that is not associated with the Golgi complex.

In the process of developing a cancer immunotherapy strategy, we have identified and characterized a novel intracellular reservoir of CD86 protein in peripheral blood monocytes. This observation emerged from studies aimed at using retrovirus vectors to genetically modify tumor cells to express the costimulatory proteins CD80 and CD86. Retrovirus-mediated expression of CD80 and CD86 in T lymphoblastoid CEM cells resulted in an unexpected intracellular focal concentration of both proteins in the genetically modified cells. By extending these studies to an analysis of CD80 and CD86 expression in PBMC, we observed that endogenous CD86 expression in peripheral blood monocytes also involved a similar intracellular focal concentration of the protein. The intracellular concentration of CD86 in monocytes was not due to storage within the Golgi apparatus, and required intact microtubules to retain structural integrity. Furthermore, as the intensity of CD86 fluorescence increased on monocytes as a function of time in vitro, the intracellular focal concentration correspondingly decreased. These results are consistent with antegrade CD86 transport from an intracellular reservoir to the cell surface membrane. In this report, we detail the intracellular and membrane localization studies with tumor cell lines and PBMC, and describe the temporal relationship between intracellular storage and trafficking of CD86 to the cell surface membrane in peripheral blood monocytes. We hypothesize that this intracellular reservoir allows rapid and sustained deployment of an important costimulatory molecule to the monocyte surface membrane during initiation and maturation of the cell-mediated immune response.

The allocation of epiblast cells to the embryonic heart and other mesodermal lineages: the role of ingression and tissue movement during gastrulation.

The cardiogenic potency of cells in the epiblast of the early primitive-streak stage (early PS) embryo was tested by heterotopic transplantation. The results of this study show that cells in the anterior and posterior epiblast of the early PS-stage embryos have similar cardiogenic potency, and that they differentiated to heart cells after they were transplanted directly to the heart field of the late PS embryo. That the epiblast cells can acquire a cardiac fate without any prior act of ingression through the primitive streak or movement within the mesoderm suggests that neither morphogenetic event is critical for the specification of the cardiogenic fate. The mesodermal cells that have recently ingressed through the primitive streak can express a broad cell fate that is characteristic of the pre-ingressed cells in the host when they were returned to the epiblast. However, mesoderm cells that have ingressed through the primitive streak did not contribute to the lateral plate mesoderm after transplantation back to the epiblast, implying that some restriction of lineage potency may have occurred during ingression. Early PS stage epiblast cells that were transplanted to the epiblast of the mid PS host embryos colonised the embryonic mesoderm but not the extraembryonic mesoderm. This departure from the normal cell fate indicates that the allocation of epiblast cells to the mesodermal lineages is dependent on the timing of their recruitment to the primitive streak and the morphogenetic options that are available to the ingressing cells at that instance.

Tropomyosin localization reveals distinct populations of microfilaments in neurites and growth cones.

The functional and structural differences between neurites and growth cones suggests the possibility that distinct microfilament populations may exist in each domain. Tropomyosins are integral components of the actin-based microfilament system. Using antibodies which detect three different sets of tropomyosin isoforms, we found that the vast majority of tropomyosin was found in a microfilament-enriched fraction of cultured cortical neurons, therefore enabling us to use the antisera to evaluate compositional differences in neuritic and growth cone microfilaments. An antibody which reacts with all known nonmuscle isoforms of the alpha Tms gene (Tm5NM1-4) stains both neurites and growth cones, whereas a second antibody against the isoform subset, Tm5NM1-2, reacts only with the neurite. A third antibody which reacts with the Tm5a/5b isoforms encoded by a separate gene from alpha Tms was strongly reactive with both neurites and growth cones in 16-h cultures but only with the neurite shaft in 40-h cultures. Treatment of neurons with cytochalasin B allowed neuritic Tm5NM1-2 to spread into growth cones. Removal of the drug resulted in the disappearance of Tm5NM1-2 from the growth cone, indicating that isoform segregation is an active process dependent on intact microfilaments. Treatment of 40-h cultures with nocodazole resulted in the removal of Tm5NM1-2 from the neurite whereas Tm5a/5b now spread back into the growth cone. We conclude that the organization of Tm5NM1-2 and Tm5a/5b in the neurite is at least partially dependent on microtubule integrity. These results indicate that tropomyosin isoforms Tm5NM1-2, Tm5NM3-4, and Tm5a/5b mark three distinct populations of actin filaments in neurites and growth cones. Further, the composition of microfilaments differs between neurites and growth cones and is subject to temporal regulation.

Epigenetic factors controlling the development of avian Purkinje neurons.

Using a unique protocol, we have developed an avian neuron culture system in which a high yield of Purkinje neurons is obtained more readily than with pre-existing methods. Purkinje neurons were identified in vitro using the specific antibodies calbindin and cyclic GMP-kinase. Survival of Purkinje neurons was dependent on astrocyte contact and enhanced by astrocytic factors supplied to the medium by a monolayer of astrocytes grown on coated membranes suspended in the culture wells but not in contact with the neurons. The age of the cerebellum from which astrocytes were obtained was shown to affect the morphological development of the Purkinje neurons suggesting the developmentally-regulated expression of growth factors. However, in the presence of the astrocytes, Purkinje neurons could only progress to a limited stage of development based on morphological criteria. The addition to the culture of cerebellar granule neurons at a time of Purkinje neuron development that they would expect to encounter them in vivo resulted in a shift of Purkinje neurons to a mature phenotype. This maturation effect was increased in response to increasing levels of granule neurons, but was independent of the granule neuron ages used. This system offers significant advantages over other Purkinje neuron culture systems and will be useful for studying the extrinsic factors involved in Purkinje neuron development and histogenesis.

Differential induction and intracellular localization of SCG10 messenger RNA is associated with neuronal differentiation.

The differentiation of neurons involves the establishment of distinct molecular compartments which regulate neuronal shape and function. This requires targeting of specific gene products to growth-associated regions of the neuron. We have investigated the temporal and spatial regulation of SCG10 gene expression during neuronal differentiation. There are two SCG10 messenger RNAs, 1 and 2 kg in length, which encode the same growth-associated protein. These messenger RNAs were found to be differentially regulated during the onset of neurite outgrowth in early rat cerebellum development. In PC12 cells, the two SCG10 messenger RNAs were shown to be differentially induced by nerve growth factor. Regulation of the 2 kb messenger RNA, but not the 1 kb messenger RNA, is dependent on the differentiation of PC12 cells, indicating that post-transcriptional regulation of SCG10 expression during neurite outgrowth. Spatial regulation of the 2 kb SCG10 messenger RNA distribution during brain development was examined by in situ hybridization. The 2 kb messenger RNA was found to be localized to the neuronal pole where outgrowth was occurring, within differentiating neurons in vivo. Intracellular localization of SCG10 messenger RNA was also observed in differentiating primary cultured neurons, with the 2 kb messenger RNA transported into growing neurites during the development of neuronal polarity. In neurons which had developed polarity, the 2 kb SCG10 messenger RNA was consistently found in the cell body and axon. This study demonstrates both temporal and spatial post-transcriptional regulation of SCG10 expression which is associated with neurite outgrowth. The directed transport and positional translation of SCG10 messenger RNA provide a potential mechanism for protein targeting and the creation of molecular compartments during neuronal differentiation.

Intracellular localization of tropomyosin mRNA and protein is associated with development of neuronal polarity.

Neuronal differentiation involves extensive rearrangement of the cytoskeleton, including the actin-based microfilament system, and establishment of molecular compartments within the neuron. The intracellular distribution of tropomyosin (Tm) mRNA in vivo and in vitro has been examined and correlated with protein targetting. The mRNAs encoding two Tm isoforms were found to be differentially localized in developing neurons. Tm-5 mRNA is localized to the axonal pole of differentiating embryonic rat neurons, in contrast to TmBr-2 mRNA distribution throughout the cell body. Tm-5 mRNA is transported into the axon of differentiating primary cultured neurons. This mRNA localization is developmentally regulated and correlates with the targeting of Tm-5 protein to growing axons. Tm-5 colocalizes with a subset of neuronal microfilaments associated with the initiation and maintenance of outgrowth. The segregation of Tm-5 is the earliest known marker of neuronal polarity and may play a role in the establishment of polarity.

Induction of neuron-specific tropomyosin mRNAs by nerve growth factor is dependent on morphological differentiation.

We have examined the expression of brain-specific tropomyosins during neuronal differentiation. Both TmBr-1 and TmBr-3 were shown to be neuron specific. TmBr-1 and TmBr-3 mRNA levels increased during the most active phase of neurite outgrowth in the developing rat cerebellum. In PC12 cells stimulated by nerve growth factor (NGF) to differentiate to the neuronal phenotype, TmBr-1 and TmBr-3 levels increased with an increasing degree of morphological differentiation. Induction of TmBr-1 and TmBr-3 expression only occurred under conditions where PC12 cells were permitted to extend neurites. NGF was unable to maintain levels of TmBr-1 and TmBr-3 with the loss of neuronal phenotype by resuspension of differentiated PC12 cells. The unique cellular expression and regulation in vivo and in vitro of TmBr-1 and TmBr-3 strongly suggests a critical role of these tropomyosins in neuronal microfilament function. The findings reveal that the induction and maintenance of the neuronal tropomyosins is dependent on morphological differentiation and the maintenance of the neuronal phenotype.

Effect of zinc on calmodulin-stimulated protein kinase II and protein phosphorylation in rat cerebral cortex.

The effect of increasing concentrations of Zn2+ (1 microM-5 mM) on protein phosphorylation was investigated in cytosol (S3) and crude synaptic plasma membrane (P2-M) fractions from rat cerebral cortex and purified calmodulin-stimulated protein kinase II (CMK II). Zn2+ was found to be a potent inhibitor of both protein kinase and protein phosphatase activities, with highly specific effects on CMK II. Only one phosphoprotein band (40 kDa in P2-M phosphorylated under basal conditions) was unaffected by addition of Zn2+. The vast majority of phosphoprotein bands in both basal and calcium/calmodulin-stimulated conditions showed a dose-dependent inhibition of phosphorylation, which varied with individual phosphoproteins. Two basal phosphoprotein bands (58 and 66 kDa in S3) showed a significant stimulation of phosphorylation at 100 microM Zn2+ with decreased stimulation at higher concentrations, which was absent by 5 mM Zn2+. A few Ca2+/calmodulin-stimulated phosphoproteins in P2-M and S3 showed biphasic behavior; inhibition at less than 100 microM Zn2+ and stimulation by millimolar concentrations of Zn2+ in the presence or absence of added Ca2+/calmodulin. The two major phosphoproteins in this group were identified as the alpha and beta subunits of CMK II. Using purified enzyme, Zn2+ was shown to have two direct effects on CMK II: an inhibition of Ca2+/calmodulin-stimulated autophosphorylation and substrate phosphorylation activity at low concentrations and the creation of a new Zn(2+)-stimulated, Ca2+/calmodulin-independent activity at concentrations of greater than 100 microM that produces a redistribution of activity biased toward autophosphorylation and an alpha subunit with an altered mobility on sodium dodecyl sulfate-containing gels.

Purification and characterization of calmodulin-stimulated protein kinase II from two-day and adult chicken forebrain.

Soluble calmodulin-stimulated protein kinase II has been purified from 2-day and adult chicken forebrain. At both ages the holoenzyme eluted from a Superose-6B column with an apparent molecular weight of approximately 700,000 daltons and contained three subunits. The subunits were found to be the counterparts of the alpha, beta, and beta' subunits of the enzyme purified from adult rat brain in that they had one-dimensional phosphopeptide maps that were indistinguishable from those of the corresponding subunit in the rat enzyme and they migrated in SDS-polyacrylamide gels with the same apparent molecular weights. However, the doublet formed by the beta subunit was much more clearly resolved in the chicken enzyme and the beta' subunit, which was much more abundant in the adult chicken than in the adult rat, was also found to be a doublet. The ratio of the concentrations of the alpha and beta subunits changed during development. By autoradiography following autophosphorylation, the alpha:beta ratios of the 2-day and adult enzymes were 0.89 +/- 0.07 and 1.92 +/- 0.26, respectively; by silver staining the alpha:beta ratios were 0.95 +/- 0.11 and 1.85 +/- 0.17, respectively. The concentration of the beta' subunit was equal to that of the beta subunit at both ages. Autophosphorylation produced a decrease in the electrophoretic mobility of the alpha and beta subunits in SDS-polyacrylamide gels and a marked decrease in the calcium dependence of the substrate phosphorylation activity of the enzyme at both ages. The purified enzyme from chicken brain appeared to be more stable under standard in vitro assay conditions than the rat enzyme, and this was particularly so for the enzyme from 2-day forebrain.

Developmental changes in protein phosphorylation in chicken forebrain. I. cAMP-stimulated phosphorylation.

The net level of cyclic AMP-stimulated protein phosphorylation was investigated in cytosolic and membrane fractions from chicken forebrain between embryonic day 13 (E13) and 52 days post-hatching. Throughout this period the majority of the net level of cAMP-stimulated phosphorylation of endogenous proteins was in the cytosolic fractions. Between day -8 (E13) and adult, the net level of cAMP-stimulated phosphorylation of endogenous proteins in the cytosol (S3) and crude synaptic plasma membrane (P2-M) fractions fell by 3 and 4 fold, respectively, when expressed per mg protein and rose by 5 and 10 fold, respectively, when expressed per fraction. The changes in specific activity were completed by 6-15 days post-hatching. The occluded cytosol (P2-S) fraction showed little change in the net level of cAMP-stimulated phosphorylation of endogenous proteins per mg protein. Major changes in phosphoprotein patterns involving both decreases and increases in phosphorylation occurred in all fractions from day -8 (E13) to day 6 post-hatch; thereafter the phosphoprotein bands and their relative intensities were unchanged. Three bands (P90 in S3; P41 and P31 in P2-M) contained major cAMP-stimulated phosphoproteins in embryonic brain but were absent after hatching. When cAMP-stimulated phosphorylation activity was measured in S3 and P-2M using an exogenous peptide substrate (Kemptide) there was no change in kinase activity per mg protein between day -8 (E13) and 30 days post-hatch. This suggests that the decrease in the net level of cAMP stimulated phosphorylation of endogenous proteins was due to the decrease in levels of endogenous phosphoproteins rather than protein kinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental changes in protein phosphorylation in chicken forebrain. II. Calmodulin stimulated phosphorylation.

The development of calmodulin stimulated protein phosphorylation, with particular reference to calmodulin-stimulated protein kinase II (CMK II), was investigated in 3 subcellular fractions of chicken forebrain: cytosol (S3), crude synaptic plasma membranes (P2-M) and occluded cytosol (P2-S). Changes in the level of calmodulin-stimulated phosphorylation of endogenous proteins occurred over a protracted time course and were not complete until after day 52 post-hatching. By day 15 post-hatching, calmodulin-stimulated phosphoproteins characteristic of embryonic fractions had all disappeared and those characteristic of adult tissue were present but not necessarily at their mature levels. The levels of CMK II were estimated from the autophosphorylation of the alpha-subunit which was the only phosphoprotein present at 53,000 Da in the 3 fractions. Overall, calmodulin-stimulated phosphorylation and CMK II levels were low in embryonic brain and high in adult brain but two specific changes in CMK II were observed during development: (1) although CMK II concentrations increased in both membrane and cytosolic fractions until day 23 the kinase was predominantly cytoplasmic (approximately 75%) until day 23, after which it became increasingly membrane bound so that by day 52 post-hatching the majority of CMK II was present in the synaptic membrane fraction, and (2) the relative concentrations of the alpha- and beta-subunits changed from an alpha:beta-value of approximately 1:1 in the 19 day embryo to approximately 1:2 by 15 days post-hatch after which no further change was seen. The occurrence of major changes in the calmodulin stimulated protein phosphorylation system for up to 6-8 weeks after synapse formation is completed in the forebrain, provides further support for the existence of a synapse maturation phase of neuronal differentiation which is distinct from synapse formation. This phase involves only a specific subset of the developmental changes occurring in the calmodulin-stimulated phosphorylation system.

Active site-directed inhibition of Ca2+/calmodulin-dependent protein kinase type II by a bifunctional calmodulin-binding peptide.

The activation of Ca2+/calmodulin (CaM)-dependent protein kinase II (CaM-KII) by Ca2+/CaM results in autophosphorylation and the generation of Ca2+/CaM-independent enzyme activity. We postulated that CaM binding and subsequent autophosphorylation alters the conformation of CaM-KII and exposes its substrate-binding and catalytic site(s). Previous peptide mapping studies on CaM-KII demonstrated the close proximity of CaM-binding and autophosphorylation domains. Analyses of the deduced amino acid sequences encoding CaM-KII have allowed the identification of its CaM-binding domain and have revealed two consensus phosphorylation sites that flank this regulatory domain. We report herein the distinct properties of two synthetic peptides modeled after the CaM-binding domain of CaM-KII. The first peptide binds CaM in a Ca2+-dependent manner and is an antagonist of CaM-KII activation (IC50 approximately equal to 75 nM). It does not, however, inhibit CaM-KII activity. A second peptide containing the same CaM-binding domain plus a putative autophosphorylation sequence at its N terminus displayed bifunctional regulatory properties. In addition to being a CaM antagonist, the latter was a potent inhibitor of Ca2+/CaM-independent kinase activity (IC50 approximately equal to 2 microM). We suggest that this bifunctional peptide represents an active site-directed inhibitory element of CaM-KII. The separation of CaM antagonist and active site-directed inhibitory properties of this peptide distinguishes CaM-KII from other CaM-dependent enzymes in which bifunctional regulatory properties appear to reside in the same peptide domain. These results indicate that the definition of site-directed inhibitory peptides should, in some cases, be expanded to include bona fide phosphorylation sites.