Natural N-terminal fragments of brain abundant myristoylated protein BASP1.

BASP1 (also known as CAP-23 and NAP-22) is a novel myristoylated calmodulin-binding protein, abundant in nerve terminals. It is considered as a signal protein participating in neurite outgrowth and synaptic plasticity. BASP1 is also present in significant amounts in kidney, testis, and lymphoid tissues.

RasGAP-associated endoribonuclease G3Bp: selective RNA degradation and phosphorylation-dependent localization.

Mitogen activation of mRNA decay pathways likely involves specific endoribonucleases, such as G3BP, a phosphorylation-dependent endoribonuclease that associates with RasGAP in dividing but not quiescent cells. G3BP exclusively cleaves between cytosine and adenine (CA) after a specific interaction with RNA through the carboxyl-terminal RRM-type RNA binding motif. Accordingly, G3BP is tightly associated with a subset of poly(A)(+) mRNAs containing its high-affinity binding sequence, such as the c-myc mRNA in mouse embryonic fibroblasts.

Phosphorylation of AZT-resistant human immunodeficiency virus type 1 reverse transcriptase by casein kinase II in vitro: effects on inhibitor sensitivity.

Casein kinase II (CKII) phosphorylates wild-type (WT) recombinant reverse transcriptase (RT) mainly in the p66 subunit in vitro. Phosphorylation of T215F RT and D67N/K70R/T215F/K219Q RT (AZT-resistant RT) in vitro increases discrimination against AZTTP 2. 5- and 3.

Detection of nucleotide- and F-actin-induced movements in the switch II helix of the skeletal myosin using its differential oxidative cleavage mediated by an iron-EDTA complex disulfide-linked to the strong actin binding site.

We have synthesized the mixed disulfide, S-(2-nitro-5-thiobenzoic acid) cysteaminyl-EDTA, using a rapid procedure and water-soluble chemistry. Its disulfide-thiol exchange reaction with rabbit myosin subfragment-1 (S-1), analyzed by spectrophotometry, ATPase assays, and peptide mapping, led to the incorporation of the cysteaminyl-EDTA group into only Cys 540 on the heavy chain and into the unique cysteine on the alkali light chains. The former thiol, residing in the strong actin binding site, reacted at a much faster rate with a concomitant 3-fold decrease in the V(max) for acto-S-1 ATPase but without change in the essential enzymatic functions of S-1.

A novel phosphorylation-dependent RNase activity of GAP-SH3 binding protein: a potential link between signal transduction and RNA stability.

A potential p120 GTPase-activating protein (RasGAP) effector, G3BP (RasGAP Src homology 3 [SH3] binding protein), was previously identified based on its ability to bind the SH3 domain of RasGAP. Here we show that G3BP colocalizes and physically interacts with RasGAP at the plasma membrane of serum-stimulated but not quiescent Chinese hamster lung fibroblasts. In quiescent cells, G3BP was hyperphosphorylated on serine residues, and this modification was essential for its activity.

Phosphorylation of an N-terminal motif enhances DNA-binding activity of the human SRY protein.

Of the several strategies that eukaryotes have evolved to modulate transcription factor activity, phosphorylation is regarded as one of the major mechanisms in signal-dependent transcriptional control. To conclusively demonstrate that the human sex-determining gene SRY is affected by such a post-translational control mechanism, we have analyzed its phosphorylation status in living cells. In the present study, we show that the cyclic AMP-dependent protein kinase (PKA) phosphorylates the human SRY protein in vitro as well as in vivo on serine residues located in the N-terminal part of the protein.

The BASP1 family of myristoylated proteins abundant in axonal termini. Primary structure analysis and physico-chemical properties.

Proteins BASP1 and GAP-43/B-50, which are abundant in nerve endings, show a number of similar physico-chemical properties. Nevertheless, they belong to different protein families. In this work, complete amino acid sequences of bovine BASP1 and human BASP1 were established.

Involvement of the Ca2+/calmodulin-dependent protein kinase II pathway in the Ca2+-mediated regulation of the capacitative Ca2+ entry in Xenopus oocytes.

Activation of the phosphoinositide transduction pathway induces capacitative Ca2+ entry in Xenopus oocytes. This can also be evoked by intracellular injection of Ins(1,4.5)P3, external application of thapsigargin and/or incubation in a Ca2+-free medium.

Analysis of the ion binding sites of calmodulin by electrospray ionization mass spectrometry.

The binding of Ca2+ and Mg2+ to four calmodulins (SynCaM 1, SynCaM 8, SynCaM 12A, and SynCaM 18A) has been studied by ESI-MS. The mass spectra were recorded by dissolving the apoproteins in methanol/water (20/80, v/v) containing 1 mM CaCl2 or 1 mM MgCl2 and the pH adjusted to 6.0 with ammonia.

p40MO15 associates with a p36 subunit and requires both nuclear translocation and Thr176 phosphorylation to generate cdk-activating kinase activity in Xenopus oocytes.

p40MO15, a cdc2-related protein, is the catalytic subunit of the kinase (CAK, cdk-activating kinase) responsible for Thr161/Thr160 phosphorylation and activation of cdk1/cdk2. We have found that strong overexpression of p40MO15 only moderately increases CAK activity in Xenopus oocytes, indicating that a regulatory CAK subunit (possibly a cyclin-like protein) limits the ability to generate CAK activity in p40MO15 overexpressing oocytes. This 36 kDa subunit was microsequenced after extensive purification of CAK activity.

Elongation factor EF-1 delta, a new target for maturation-promoting factor in Xenopus oocytes.

A new physiological target for Cdc2 protein kinase has been identified. It corresponds to a protein EF-1 delta, a constituent of the nucleotide exchange factor EF-1 beta gamma delta, involved in the elongation step of protein synthesis. EF-1 delta is phosphorylated by Cdc2 kinase on threonine and serine residues.

Activation of p34cdc2 protein kinase by microinjection of human cdc25C into mammalian cells. Requirement for prior phosphorylation of cdc25C by p34cdc2 on sites phosphorylated at mitosis.

Human cdc25C protein, a specific tyrosine phosphatase that activates the p34cdc2 protein kinase at mitosis, is itself a phosphoprotein that shows increased phosphorylation during the G2-M transition. In vitro, cdc25C protein is substantially phosphorylated by purified p34cdc2-cyclin B protein kinase. Of seven putative phosphorylation sites for p34cdc2 protein kinase present in human cdc25C, five are phosphorylated by p34cdc2 protein kinase in vitro, as assessed by tryptic phosphopeptide mapping and peptide sequencing.

Definition of the EGTA-independent interface involved in the serum gelsolin-actin complex.

The gelsolin-actin complex in the presence of Ca2+ revealed at least three interacting sites on the gelsolin molecule located in the S1, S2-3, and S4-6 domains. In the presence of EGTA, the N-terminal domain of gelsolin is known to be involved. However, the corresponding site on the surface of actin is poorly defined.

The MO15 gene encodes the catalytic subunit of a protein kinase that activates cdc2 and other cyclin-dependent kinases (CDKs) through phosphorylation of Thr161 and its homologues.

Phosphorylation of Thr161, a residue conserved in all members of the cdc2 family, has been reported to be absolutely required for the catalytic activity of cdc2, the major regulator of eukaryotic cell cycle. In the present work, we have purified from starfish oocytes a kinase that specifically activates cdc2 in a cyclin-dependent manner through phosphorylation of its Thr161 residue. Our most highly purified preparation contained only two major proteins of apparent M(r) 37 and 40 kDa (p37 and p40), which could not be separated from each other without loss of activity.

Photochemical cross-linking of the skeletal myosin head heavy chain to actin subdomain-1 at Arg95 and Arg28.

F-actin specifically substituted with the photocross-linker, p-azidophenylglyoxal, at Arg95 and Arg28 was isolated and characterized. Upon complexation with myosin subfragment-1 (S1) and photolysis at 365 nm, it was readily cross-linked to the S1 heavy chain with a yield of about 13-25%, generating four major actin-heavy-chain adducts with molecular masses in the range 165-240 kDa. The elevated Mg(2+)-ATPase of the covalent complexes displayed a turnover rate of 33 +/- 8 s-1 which is similar to the values reported earlier for other acto-S1 conjugates.

Influence of Ca-activated brevin on the mechanical properties of skinned smooth muscle.

Solutions of purified brevin were applied to skinned thin bundles or isolated fibres of smooth muscle. This produced a sharp drop of isometric tension, an effect due to the severing effect of brevin on actin filaments, partially depleted from tropomyosin in skinned preparations. On skinned single fibres, brevin accelerates the speed of unloaded shortening.

Dephosphorylation of cdc2 on threonine 161 is required for cdc2 kinase inactivation and normal anaphase.

Exit from metaphase of the cell cycle requires inactivation of MPF, a stoichiometric complex between the cdc2 catalytic and the cyclin B regulatory subunits, as well as that of cyclin A-cdc2 kinase. Inactivation of both complexes depends on proteolytic degradation of the cyclin subunit, yet cyclin proteolysis is not sufficient to inactivate the H1 kinase activity of cdc2. Genetic evidence strongly suggests that type 1 phosphatase plays a key role in the metaphase-anaphase transition of the cell cycle.

Complex stiffness of smooth muscle cytoplasm in the presence of Ca-activated brevin.

Brevin, an F-actin severing protein, regulates actin gel-sol transformation in a Ca(2+)-dependent way. Here, we tested its effect on the stiffness of the cytoplasm of skinned smooth muscle, in the absence of actin-myosin interaction (inhibited myosin ATPase). Complex stiffness was measured by imposing sinusoidal stretches and releases at different frequencies (1-50 Hz).

Cellular mechanism of metformin action.

Activation of the insulin receptor tyrosine kinase and tyrosine phosphorylation of intracellular substrates are important steps in insulin signalling. In order to elucidate the cellular mechanism of action of metformin (NN'dimethylbiguanide) we have focused towards the effects of metformin on the insulin receptor kinase, the phosphorylation cascade and the biological effect of insulin. Since annexins (lipocortins) have been recently recognized as substrates of several tyrosine kinases we have investigated the effect of metformin on phosphorylation of annexins after insulin stimulation or microinjection of pp60c-src kinase in Xenopus laevis oocytes.

Identification and tissue localization of an eosinophil 17 kDa protein accumulating in rat uterus upon estradiol treatment.

In a previous paper (J. Steroid Biochem. 29 (1988) 475-480), the isolation of a 17 kDa protein that was dramatically induced in the uterus of estrogen-treated spayed rats was presented.

The action of brevin, an F-actin severing protein, on the mechanical properties and ATPase activity of skinned smooth muscle.

Brevin is a protein which regulates the actin gel-sol transformation: it severs F-actin filaments into shorter ones. This action is Ca-dependent and is prevented by tropomyosin. We tested the effect of brevin on isometric contractions of skinned smooth muscle (taenia coli) and noted a dramatic loss of tension that possibly reflects some F-actin fragmentation.

Ontogenesis of rabbit liver cytochrome P450. Evidence for a cytochrome P450-IIE (3a)-related form prevailing during the post-natal period.

The liver hydroxylating system, mainly composed of cytochromes P450, is not highly active during foetal life. If develops after birth and reaches the adult level several weeks post-partum. We have studied the ontogenesis of rabbit cytochrome P450 during the post-natal period.

High levels of HMG1-2 protein expression in the cytoplasm and nucleus of hydrocortisone sensitive amphibian thymocytes.

A major 26 kDa protein was identified in the cytoplasmic and nuclear compartments of axolotl thymocytes. A polyclonal antiserum was produced against the denatured form of this protein. High levels of 26 kDa were expressed by hydrocortisone-sensitive lymphocytes which represent a major thymocyte subpopulation in young animals.

Involvement of protein phosphatases 1 and 2A in the control of M phase-promoting factor activity in starfish.

Specific inhibition of types 1 and 2A protein phosphatases by microinjection of okadaic acid (OA) into starfish oocytes induced germinal vesicle breakdown and activation of M phase-promoting factor (MPF) and histone H1 kinase. The effects were evident in immature oocytes arrested at first meiotic prophase as well as in fully mature oocytes arrested at the pronucleus stage. In addition, MPF and histone H1 kinase were stabilized for several hours and protected from inactivation by inhibition of type 1 protein phosphatases with either OA or specific anti-phosphatase antibodies.