Endogenous Kinase Activity Research Articles

A signal transduction pathway for activation of the mdr1 promoter involves the proto-oncogene c-raf kinase

To investigate the regulation of expression of the human mdr1 gene, the response of the mdr1 promoter to signals involved in cell proliferation was examined. The activity of the mdr1 promoter was measured in transiently transfected NIH 3T3 cells, which were stimulated to enter the cell cycle by addition of serum, platelet-derived growth factor, or transforming growth factor alpha. Addition of serum to quiescent NIH 3T3 cells resulted in a 6-8-fold activation of mdr1 promoter activity, which peaked at 10 h, just prior to S-phase. Treatment of serum-starved cells with the serum mitogens platelet-derived growth factor and transforming growth factor alpha also activated the mdr1 promoter. To define components of the signal cascade resulting in mdr1 promoter activation, the role of c-Raf kinase, a serine/threonine kinase important in mitogen-activated signal transduction, was examined. We measured the effects of a constitutively activated Raf kinase, v-raf, and a dominant negative Raf mutant, c-Raf-C4, on mdr1 promoter activity in NIH 3T3 and HepG2 cells. In serum-starved NIH 3T3 cells, v-raf increased mdr1 promoter activity approximately 10-fold compared to a v-raf frame-shift control. Raf responsiveness of the mdr1 promoter was localized to sequences between -69 and -41, relative to the initiation site. Serum stimulation of the mdr1 promoter was blocked by co-transfection of NIH 3T3 cells with the dominant negative Raf mutant c-Raf-C4. In the human hepatoma cell line HepG2, which has high endogenous Raf kinase activity (Bruder, J. T., Heidecker, G., and Rapp, U. R. (1992) Genes & Dev. 6, 545-556), co-transfection with c-Raf-C4 decreased mdr1 promoter activity 20-fold. Taken together, these data suggest that the mdr1 gene is transcriptionally regulated by normal cellular signaling events involving activation of c-Raf kinase.

Inhibition of bradykinin- and thapsigargin-induced Ca2+ entry by tyrosine kinase inhibitors

We examined the involvement of tyrosine kinase activity in the bradykinin (BK)-mediated signal transduction process. Immunoblots with anti-phosphotyrosine antibodies following BK stimulation of human fibroblasts showed tyrosine phosphorylation of specific proteins that could be inhibited by the tyrosine kinase inhibitors genistein and tyrphostin. Image analysis data from individual cells stimulated by BK in the presence of genistein and tyrphostin showed that these inhibitors block the plateau phase but not the rapid transient phase of the BK-induced calcium response. That the loss of the plateau phase was due to blockage of calcium entry rather than stimulation of calcium pump activity was confirmed by examining the influx of Ba2+ following BK stimulation. The Ca2+ imaging results were confirmed by 45Ca2+ uptake measurements and extended to another tyrosine kinase inhibitor (methyl 2,5-dihydroxycinnamate), which was found to interfere with the fura-2 signal and therefore could not be used in imaging experiments. The fact that three structurally distinct inhibitors of tyrosine kinase activity inhibited BK-stimulated calcium influx, while an inactive analogue of genistein (daidzein) did not, strongly suggests the involvement of tyrosine kinases in the regulation of a BK-induced calcium entry pathway. To our knowledge, this is the first report of tyrosine kinase involvement in the regulation of calcium entry following activation of a receptor that lacks endogenous tyrosine kinase activity and is known to be coupled to phosphatidylinositol turnover. We found that calcium entry in HSWP (human foreskin fibroblast) cells can also be stimulated by emptying the intracellular calcium stores with thapsigargin. Genistein also inhibits the plateau phase of the thapsigargin-induced calcium response while leaving the transient phase intact. This suggests that the Ca2+ influx pathway induced by depletion of intracellular calcium stores with thapsigargin also may be regulated via a tyrosine kinase pathway.

Raf-1 kinase is essential for early Xenopus development and mediates the induction of mesoderm by FGF

Animal cap explants from Xenopus embryos injected with a dominant negative Raf-1 mutant, termed NAF (not a functional Raf), demonstrated a complete block to basic fibroblast growth factor (FGF)-stimulated mesoderm induction. Activin induction of mesoderm was normal in embryos that expressed NAF. Injection of NAF RNA into 2-cell stage embryos blocked normal development during neurula stages and caused severe posterior truncations in tadpoles. The phenotype induced by NAF could be rescued by coinjection of wild-type raf-1 RNA. The NAF mutant functioned by specifically blocking the activation of endogenous Raf kinase activity. These findings suggest that Raf-1 mediates FGF, but not activin, receptor signaling during mesoderm induction and implicate Raf-1 as a key signaling molecule in the development of posterior structure.

Study of phosphorylation of translation elongation factor 2 (EF-2) from wheat germ

Phosphorylation of elongation factor 2 (EF-2) by specific Ca 2+/calmodulin-dependent kinase is considered as a possible mechanism of regulation of protein biosynthesis in animal cells at the level of polypeptide chain elongation. In this report we show that wheat germ EF-2 can be intensively phosphorylated by the rabbit reticulocyte EF-2 kinase. Phosphorylation results in inhibition of the activity of plant EF-2 in poly(U)-dependent cell-free translation system. Thus, the activity of EF-2 in plant cells can be potentially regulated by phosphorylation. However, we could not detect endogenous EF-2 kinase activity in wheat germ either in vitro or in vivo. Furthermore, EF-2 kinase activity is not displayed in different organs of wheat and other higher plants.

Negative Regulation of G1 in Mammalian Cells: Inhibition of Cyclin E-Dependent Kinase by TGF-β

Transforming growth factor-beta (TGF-beta) is a naturally occurring growth inhibitory polypeptide that arrests the cell cycle in middle to late G1 phase. Cells treated with TGF-beta contained normal amounts of cyclin E and cyclin-dependent protein kinase 2 (Cdk2) but failed to stably assemble cyclin E-Cdk2 complexes or accumulate cyclin E-associated kinase activity. Moreover, G1 phase extracts from TGF-beta-treated cells did not support activation of endogenous cyclin-dependent protein kinases by exogenous cyclins. These effects of TGF-beta, which correlated with the inhibition of retinoblastoma protein phosphorylation, suggest that mammalian G1 cyclin-dependent kinases, like their counterparts in yeast, are targets for negative regulators of the cell cycle.

Cyclic AMP-mediated Phosphorylation and Insolubilization of a 49-kDa Cytoskeletal Marker Protein of Lens Fiber Terminal Differentiation

Terminal differentiation of chick lens fiber cells has been previously characterized by the accumulation, acidification via phosphorylation, and increased membrane association of a 49-kDa cytoskeletal protein. In these studies, we examine: (1) the subcellular distribution of the 49-kDa protein with regard to ageing and isoform composition; and (2) potential mechanisms regulating 49-kDa phosphorylation and insolubilization. With conventional Western blotting techniques, the 49-kDa protein is found exclusively in insoluble form within terminally differentiated nuclear fiber cells. Cortical fibers, on the other hand, exhibit a more widespread subcellular distribution of the 49-kDa protein. On two-dimensional gels, cortical 49-kDa isoelectric variants segregate according to their ease of sedimentation. After homogenization in detergent-containing buffers, the major isoform of the 49-kDa protein found in low speed pellets (40 000 g, 20 min) exhibits an acidic pI. The 40 000 g supernate and the high speed pellet (100 000 g, 2 hr) which is sedimented from this supernate are enriched in more basic isoforms of the 49-kDa protein. The 100 000 g supernate overlying the high speed pellet is dominated by the most basic isoform. With in vitro phosphorylation assays, the 49 kDa protein is shown to be a major substrate affected by endogenous cAMP-dependent mechanisms. Both the low and high speed pellets exhibit endogenous cAMP-dependent kinase activity. An inhibitor of cAMP-dependent protein kinase activity is also found in soluble lens fractions. Conversion of the 49-kDa protein into more acidic, phosphorylated isoforms increases its insolubility and ease of sedimentation. The latter results exactly mimic the normal processing of the 49-kDa protein during lens fiber development and provide a basis for future studies examining intracellular mechanisms involved in regulating lens differentiation.

Characterization of the endogenous protein kinase activity of the hepatitis B virus.

During the assembly of the nucleocapsid of the hepatitis B virus a protein kinase, probably of cellular origin, is encapsidated. This enzyme phosphorylates serine residue(s) localized within the lumen of the particle. By using purified, liver-derived core particles, we characterized the protein kinase activity in the presence of different ions and inhibitors. Controls were performed with cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) and recombinant core particles. We showed that the endogenous protein kinase of the core particles was not inhibited by H89, a specific inhibitor of PKA. Staurosporine, a selective inhibitor of PKC inhibited the endogenous kinase activity only within the first minutes of the reaction. In contrast, quercetine, a selective inhibitor of the protein kinase M (PKM) did not inhibit during the first minutes but inhibited efficiently during later phases of incubation. PKM represents an enzymatically active proteolytic fragment of PKC. These results suggest that PKC is encapsidated into human core particles and is converted to PKM during the in vitro reaction. This conclusion implies the association of a protease activity localized with the HBV nucleocapsid inside liver-derived core particles.

Effects of GTP analogues and activation of endogenous protein kinases on photoaffinity labeling with [ 3H](+)PN200-110 of crude membranes from rat heart and brain

The effects of GTP analogues and conditions in which various endogenous protein kinases were activated on photoaffinity labeling with [ 3H](+)PN200-110 (PN) of crude membranes from rat cardiac muscle and whole brain were investigated. Photoaffinity labeling with 20 nM [ 3H](+)PN of these crude membranes was decreased by 100 μM GTP-γ-S, but not by 100 μM GTP or 100 μM GDP-β-S. Similar results were obtained on the effects of GTP and its analogues on the specific binding of 20 nM [ 3H](+)PN to these crude membranes under the same conditions. Activation of endogenous protein kinases in these crude membranes did not influence the photoaffinity labeling with [ 3H](+)PN. These results suggested the binding sites, or DPH-sensitive, or L-type, calcium channels in crude membranes from rat cardiac muscle and whole brain are directly or indirectly modulated by endogenous GTP-binding protein, but not by various endogenous protein kinases in these crude membranes.

P21ras activation via hemopoietin receptors and c-kit requires tyrosine kinase activity but not tyrosine phosphorylation of p21ras GTPase-activating protein.

Products of the ras gene family, termed p21ras, are GTP-binding proteins that have been implicated in signal transduction via receptors encoding tyrosine kinase domains. Recent findings have defined a superfamily of hemopoietin receptors that includes receptors for a number of interleukins and colony-stimulating factors. The intracellular portions of these receptors show only restricted homologies, have no tyrosine kinase domain, and provide no clues to the mode of signal transduction. However, in most cases the factors stimulate tyrosine phosphorylation. We demonstrate here that ligand-induced activation of the interleukin (IL)-2, IL-3, IL-5, and granulocyte-macrophage colony-stimulating factor receptors resulted in activation of p21ras in various hemopoietic cell lines. The only cytokine tested that binds to a hemopoietin receptor and that did not activate p21ras was IL-4. Activation of p21ras was also observed in response to Steel factor, which stimulates the endogenous tyrosine kinase activity of the c-kit receptor, as well as with phorbol esters, which activate protein kinase C. Experiments with protein kinase inhibitors implicated tyrosine kinase activity, but not protein kinase C activity, as the upstream signal in p21ras activation via these growth factor receptors. Attempts to demonstrate tyrosine phosphorylation of the p21ras GTPase-activating protein (GAP) were negative, suggesting that phosphorylation of GAP may not be the major mechanism for regulation of p21ras activity by tyrosine kinases.

Isolation and characterization of rat 3Y1 fibroblast clones overexpressing the src homology region of phospholipase C-gamma 2.

To examine the regulatory function of the src-related SH2 and SH3 (SH2/SH3) region of phospholipase C-gamma 2 (PLC-gamma 2), we expressed this region of rat PLC-gamma 2 cDNA in rat 3Y1 fibroblasts and isolated and characterized a number of clones (approximately 20 clones). An increase of endogenous tyrosine kinase activity was observed in all cell clones that highly expressed a translational product of the SH2/SH3 domain. Moreover, endogenous phosphatidylinositol 4,5-bisphosphate hydrolyzing activity was also enhanced in these clones, and PLC-gamma 1 seemed to be preferentially activated among endogenous PLC isozymes. Genistein, an inhibitor of tyrosine kinase, inhibited this activation of PLC-gamma 1, and tyrosine phosphorylation was observed on PLC-gamma 1 molecules, indicating the involvement of tyrosine kinases in the PLC-gamma 1 activation. These results suggest that the SH2/SH3 region of PLC-gamma would function as a multidirectional regulator which controls at least two major signaling pathways: tyrosine kinase and phosphatidylinositol 4,5-bisphosphate hydrolysis.

Phosphoinositide Hydrolysis and Ensuing Calcium and Potassium Fluxes: Role in the Action of EGF and Other Growth Factors

Phosphoinositide hydrolysis and stimulated ion fluxes are part of the host of rapid intracellular events triggered by the activation of growth factor receptors. In the case of EGF and other factors (PDGF, FGF) addressed to receptors endowed with endogenous tyrosine kinase activity, the phospholipase C responsible for the signalling reaction is the γ1 isoform. The enzyme can bind directly to a recently identified domain of the EGF receptor when the latter undergoes autophosphorylation as a consequence of ligand binding. The bound enzyme is then phosphorylated at multiple sites, an event that markedly increases its activity. Of the second messengers generated by the hydrolysis, one, diacylglycerol, activates protein kinase C, an enzyme that mediates (among other effects) the feedback desensitization of the EGF receptor; the other messenger, inositol-1,4,5-trisphosphate, activates the release of Ca2+ from intracellular stores. Concomitantly, Ca2+ influx across the plasma membrane is stimulated via cationic, nonselective channels, and the membrane potential is increased by the activation of Ca2+-dependent K+ channels. Recent results with selective blockers of those two responses (imidazole drugs and charibdotoxin, respectively) have revealed that hyperpolarization has only a marginal role in the EGF-induced cell proliferation whereas Ca2+ influx is important since its block results in a substantial decrease of cell growth. The final part of the review concerns the role of phosphoinositide hydrolysis in the process of T lymphocyte activation. In these cells, the T cell receptor has been shown to interact with tyrosine kinases that are not directly inserted across the plasma membrane, and to activate them. The mechanism of transmembrane signalling resembles therefore (from this point of view) that of the EGF receptor. The mechanism by which changes in [Ca2+]i and of other cytoplasmatic parameters are transferred to the nucleus and stimulate proliferation are still poorly understood. The possible involvement of G proteins, such as ras, and of protein phosphatases is discussed.

Identification and characterization of the ATP.Mg-dependent protein phosphatase activator (FA) as a microtubule protein kinase in the brain.

The activating factor of ATP.Mg-dependent protein phosphatase (FA) has been identified in brain microtubules. When using purified MAP-2 (microtubule associated protein 2) and tau proteins as substrates, FA could phosphorylate MAP-2 to 16 moles of phosphates per mole of protein with a Km value of 0.4 microM, and tau proteins to 4 moles of phosphates per mole of proteins with a Km value of about 3 microM. When using microtubules as substrates, FA could enhance many-fold the endogenous phosphorylation of many microtubule-associated proteins including MAP-2, tau proteins, and several low-molecular-weight MAPs. In contrast to other reported MAP kinases, such as cAMP-dependent protein kinase and Ca+2/phospholipid-dependent protein kinase, the FA-catalyzed phosphorylation of tau proteins could cause an electrophoretic mobility shift on sodium dodecyl sulfate polyacrylamide gel electrophoresis, suggesting that a dramatic conformational change of tau proteins was produced by FA. Peptide mapping analysis of the phosphopeptides derived from SV8 protease digestion revealed that FA could phosphorylate MAP-2 and tau proteins on at least four specific sites distinctly different from those phosphorylated by cAMP-dependent and Ca+2/phospholipid-dependent MAP kinases. Quantitative analysis further indicated that approximately 19% of the total endogenous kinase activity in brain microtubules was due to FA. Taken together, the results provide initial evidence that the ATP.Mg-dependent protein phosphatase activating factor (FA) is a potent and unique MAP kinase, and may represent one of the major factors involved in phosphorylation of brain microtubules.

Structural and functional characterization of calelectrins from bovine liver.

Two Ca2(+)-dependent membrane-binding proteins with apparent molecular weights of 70000 (calelectrin70) and 32000 (calelectrin32) were isolated from bovine liver using phenyl-Sepharose affinity chromatography followed by diethylaminoethyl (DEAE)-cellulose and Ultrogel AcA44 chromatographies. Limited proteolysis and immunological analyses indicated that calelectrin32 was not a digested product from calelectrin70. Both calelectrins bound to phosphatidylserine and to calmodulin in a Ca2(+)-dependent manner. Circular dichroism studies showed that the apparent alpha-helical contents of calelectrin70 and calelectrin32 were 25 and 40%, respectively and they underwent Ca2(+)-dependent conformational changes. When the calelectrins were incubated with a brain microtubule preparation, they were phosphorylated by endogenous kinase(s) and phosphorylation occurred on serine residues. Moreover, calelectrin70 showed an inhibitory action on endogenous kinase activity in the presence of Ca2+.

No T-cell tyrosine protein kinase signalling or calcium mobilization after CD4 association with HIV-1 or HIV-1 gp120

The T lymphocyte surface protein CD4 is an integral membrane glycoprotein noncovalently associated with the tyrosine protein kinase p56lck. In normal T cells, surface association of CD4 molecules with other CD4 molecules or other T-cell surface proteins, such as the T-cell antigen receptor, stimulates the activity of the p56lck tyrosine kinase, resulting in the phosphorylation of various cellular proteins at tyrosine residues. Thus, the signal transduction in T cells generated through the surface engagement of CD4 is similar to that observed for the class of growth factor receptors possessing endogenous tyrosine kinase activity. As CD4 is also the cellular receptor for the human immunodeficiency virus (HIV), binding of the virus or gp120 (the virus surface protein responsible for specific CD4+ T-cell association) could mimic the types of immunological interactions that have previously been found to stimulate p56lck and trigger T-cell activation pathways. We have evaluated this possibility and report here that binding of HIV-1 or the virus glycoprotein gp120 to CD4+ human T cells fails to elicit detectable p56lck-dependent tyrosine kinase activation and signalling, alterations in the composition of cellular phosphotyrosine-containing proteins, or changes in intracellular Ca2+ concentration.

Accumulation of galactosylsphingosine (psychosine) does not interfere with phosphorylation and methylation of myelin basic protein in the twitcher mouse

In attempts to elucidate mechanisms of demyelination in the twitcher mouse (Twi), phosphorylation and methylation of myelin basic protein (MBP) were examined in the brainstem and spinal cord of this species. Phosphorylation of MBP in isolated myelin by an endogenous kinase and an exogenous [32P]ATP was not impaired and protein kinase C activity in the brain cytosol was not reduced. When the methylation of an arginine residue of MBP was examined in slices of the brainstem and spinal cord, using [3H]methionine as a donor of the methyl groups, no difference was found between Twi and the controls. Radioactivity of the [3H] methionine residue of MBP of Twi was also similar to that of the controls. Thus, accumulation of psychosine in Twi does not interfere with the activity of endogenous kinase, methylation of MBP, and the synthesis and transport of MBP into myelin membrane.

Nerve growth factor and fibroblast growth factor selectively activate a protein kinase that phosphorylates high molecular weight microtubule-associated proteins. Detection, partial purification, and characterization in PC12 cells.

A cell-free assay has been developed to detect and characterize a nerve growth factor (NGF)-stimulated protein kinase activity in PC12 cells that phosphorylates high molecular weight microtubule-associated proteins (HMW-MAPs). The activity was partially purified and separated from other endogenous nonregulated HMW-MAP kinase activities by chromatography on heparin-Sepharose and Mono-Q resin. Characterization of the NGF-activated kinase (designated HMK) revealed the following features. 1) Both MAP1 and MAP2 are phosphorylated with approximately equal efficiencies. 2) Activation reaches a plateau within 3 min of NGF treatment and persists for approximately 60 min; subsequently, a substantial decline occurs by 5 h. 3) Maximal activation reaches 15-20-fold; activation is nearly as high with fibroblast growth factor, an agent that mimics NGF in promoting PC12 cell neuronal differentiation. 4) Epidermal growth factor and depolarizing levels of K+ stimulate HMK activity by only 2-4-fold; additional agents without PC12 cell differentiation activity (insulin, phorbol ester, and a permeant cAMP analogue) do not stimulate HMK activity. 5) The divalent cation requirement shows a preference for Mn2+ over Mg2+. 6) There is inhibition by 10 mM 2-aminopurine but not by 6-thioguanine, heparin, or NaF. 7) HMW-MAPs and myelin basic protein are effective substrates while histones IIIs and H1, dephospho-beta-casein, and S6 protein are not phosphorylated by HMK. These and other features appear to distinguish HMK from a variety of other well-characterized protein kinases as well as from other previously described NGF-activated kinases. The properties of HMK indicate that it could play a role in the signaling pathway for growth-factor-promoted neuronal differentiation.

P29: a novel tyrosine-phosphorylated membrane protein present in small clear vesicles of neurons and endocrine cells.

A novel membrane protein from rat brain synaptic vesicles with an apparent 29,000 Mr (p29) was characterized. Using monospecific polyclonal antibodies, the distribution of p29 was studied in a variety of tissues by light and electron microscopy and immunoblot analysis. Within the nervous system, p29 was present in virtually all nerve terminals. It was selectively associated with small synaptic vesicles and a perinuclear region corresponding to the area of the Golgi complex. P29 was not detected in any other subcellular organelles including large dense-core vesicles. The distribution of p29 in various subcellular fractions from rat brain was very similar to that of synaptophysin and synaptobrevin. The highest enrichment occurred in purified small synaptic vesicles. Outside the nervous system, p29 was found only in endocrine cell types specialized for peptide hormone secretion. In these cells, p29 had a distribution very similar to that of synaptophysin. It was associated with microvesicles of heterogeneous size and shape that are primarily concentrated in the centrosomal-Golgi complex area. Secretory granules were mostly unlabeled, but their membrane occasionally contained small labeled evaginations. Immunoisolation of subcellular organelles from undifferentiated PC12 cells with antisynaptophysin antibodies led to a concomitant enrichment of p29, synaptobrevin, and synaptophysin, further supporting a colocalization of all three proteins. P29 has an isoelectric point of approximately 5.0 and is not N-glycosylated. It is an integral membrane protein and all antibody binding sites are exposed on the cytoplasmic side of the vesicles. Two monoclonal antibodies raised against p29 cross reacted with synaptophysin, indicating the presence of related epitopes. P29, like synaptophysin, was phosphorylated on tyrosine residues by endogenous tyrosine kinase activity in intact vesicles.

Early activation of endogenous pp60src kinase activity during neuronal differentiation of cultured human neuroblastoma cells.

The proto-oncogene product pp60c-src is a tyrosine-specific kinase with a still unresolved cellular function. High levels of pp60c-src in neurons and the existence of a neuronal pp60c-src variant, pp60c-srcN, suggest participation in the progress or maintenance of the differentiated phenotype of neurons. We have previously reported that phorbol esters, e.g., 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulate human SH-SY5Y neuroblastoma cells to neuronal differentiation, as monitored by morphological, biochemical, and functional differentiation markers. In this report, we describe activation of the pp60src (pp60c-src and pp60c-srcN) kinase activity observed at 6 h after induction of SH-SY5Y cells with TPA. This phenomenon coincides in time with neurite outgrowth, formation of growth cone-like structures, and an increase of GAP43 mRNA expression, which are the earliest indications of neuronal differentiation in these cells. The highest specific src kinase activity (a three- to fourfold increase 4 days after induction) was noted in cells treated with 16 nM TPA; this concentration is optimal for development of the TPA-induced neuronal phenotype. During differentiation, there was no alteration in the 1:1 ratio of pp60c-src to pp60c-srcN found in untreated SH-SY5Y cells. V8 protease and trypsin phosphopeptide mapping of pp60src from in vivo 32P-labeled cells showed that the overall phosphorylation of pp60src was higher in differentiated than in untreated cells, mainly because of an intense serine 12 phosphorylation. Tyrosine 416 phosphorylation was not detectable in either cell type, and no change during differentiation in tyrosine 527 phosphorylation was observed.

Phosphorylation of caldesmon

The phosphorylation of caldesmon was studied to determine if kinase activity reflected either an endogenous kinase or caldesmon itself. Titration of kinase activity with calmodulin yielded maximum activity at substoichiometric ratios of calmodulin/caldesmon. The sites of phosphorylation on caldesmon for calcium/calmodulin-dependent protein kinase II and endogenous kinase were the same, but distinct from protein kinase C sites. Phosphorylation in the presence of Ca 2+ and calmodulin resulted in a subsequent increase of endogenous kinase activity in the absence of Ca 2+. These results suggest that caldesmon is not a protein kinase and that kinase activity in caldesmon preparations is due to calcium/calmodulindependent protein kinase II.

A casein kinase II-related activity is involved in phosphorylation of microtubule-associated protein MAP-1B during neuroblastoma cell differentiation.

A neuroblastoma protein related to the brain microtubule-associated protein, MAP-1B, as determined by immunoprecipitation and coassembly with brain microtubules, becomes phosphorylated when N2A mouse neuroblastoma cells are induced to generate microtubule-containing neurites. To characterize the protein kinases that may be involved in this in vivo phosphorylation of MAP-1B, we have studied its in vitro phosphorylation. In brain microtubule protein, MAP-1B appears to be phosphorylated in vitro by an endogenous casein kinase II-like activity which also phosphorylates the related protein MAP-1A but scarcely phosphorylates MAP-2. A similar kinase activity has been detected in cell-free extracts of differentiating N2A cells. Using brain MAP preparations devoid of endogenous kinase activities and different purified protein kinases, we have found that MAP-1B is barely phosphorylated by cAMP-dependent protein kinase, Ca/calmodulin-dependent protein kinase, or Ca/phospholipid-dependent protein kinase whereas MAP-1B is one of the preferred substrates, together with MAP-1A, for casein kinase II. Brain MAP-1B phosphorylated in vitro by casein kinase II efficiently coassembles with microtubule proteins in the same way as in vivo phosphorylated MAP-1B from neuroblastoma cells. Furthermore, the phosphopeptide patterns of brain MAP-1B phosphorylated in vitro by either purified casein kinase II or an extract obtained from differentiating neuroblastoma cells are identical to each other and similar to that of in vivo phosphorylated neuroblastoma MAP-1B. Thus, we suggest that the observed phosphorylation of a protein identified as MAP-1B during neurite outgrowth is mainly due to the activation of a casein kinase II-related activity in differentiating neuroblastoma cells. This kinase activity, previously implicated in beta-tubulin phosphorylation (Serrano, L., J. Díaz-Nido, F. Wandosell, and J. Avila, 1987. J. Cell Biol. 105: 1731-1739), may consequently have an important role in posttranslational modifications of microtubule proteins required for neuronal differentiation.