Identification of Tetrahymena 14-nm filament-associated protein as elongation factor 1 alpha.

Tetrahymena 14-nm filament-forming protein has dual functions as a citrate synthase in mitochondria and as a cytoskeletal protein involved in oral morphogenesis and in pronuclear behavior during conjugation. By immunoblotting using monoclonal and polyclonal antibodies following two-dimensional gel electrophoresis, we demonstrated that the 14-nm filament protein fraction contained two 49-kDa proteins whose isoelectric points were 8.0 and 9.0; a monoclonal antibody (MAb) 26B4 and a polyclonal antibody 49KI reacted only to a pI 8.0 protein, while two other MAbs, 11B6 and 11B8, reacted only to a pI 9.0 protein. From the N-terminal amino acid sequences, the pI 8.0 protein was identified as the previously reported 14-nm filament-forming protein/citrate synthase, but the pI 9.0 protein N-terminal sequence had no similarity with that of the pI 8.0 protein. The pI 9.0 protein is considered to be a 14-nm filament-associated protein since the pI 9.0 protein copurifies with the pI 8.0 protein during two cycles of an assembly and disassembly purification protocol. Cloning and sequencing the pI 9.0 protein gene from a Tetrahymena pyriformis cDNA library, we identified the pI 9.0 protein as elongation factor 1 alpha (EF-1 alpha) based on it sharing 73-76% sequence identity with EF-1 alpha from several species.     

Tetrahymena 14-nm filament-forming protein has citrate synthase activity

The Tetrahymena 14-nm filament-forming protein (49K protein) is a structural protein involved in oral morphogenesis and in pronuclear behavior during conjugation. Cloning the 49K protein gene from a Tetrahymena thermophila cDNA library, we found that its primary structure exhibits a high sequence identity (51.5%) with porcine heart citrate synthase and retains functional domains. The 49K protein actually possesses citrate synthase activity, and is detected in mitochondria. These results suggest that the 49K protein has dual functions as both a respiratory enzyme and a structural protein in the cytoskeleton.     

Phosphorylation and 14-3-3 binding of Arabidopsis trehalose-phosphate synthase 5 in response to 2-deoxyglucose

Trehalose-6-phosphate is a 'sugar signal' that regulates plant metabolism and development. The Arabidopsis genome encodes trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphatase (TPP) enzymes. It also encodes class II proteins (TPS isoforms 5-11) that contain both TPS-like and TPP-like domains, although whether these have enzymatic activity is unknown. In this paper, we show that TPS5, 6 and 7 are phosphoproteins that bind to 14-3-3 proteins, by using 14-3-3 affinity chromatography, 14-3-3 overlay assays, and by co-immunoprecipitating TPS5 and 14-3-3 isoforms from cell extracts. GST-TPS5 bound to 14-3-3s after in vitro phosphorylation at Ser22 and Thr49 by either mammalian AMP-activated protein kinase (AMPK) or partially purified plant Snf1-related protein kinase 1 (SnRK1s). Dephosphorylation of TPS5, or mutation of either Ser22 or Thr49, abolished binding to 14-3-3s. Ser22 and Thr49 are both conserved in TPS5, 7, 9 and 10. When GST-TPS5 was expressed in human HEK293 cells, Thr49 was phosphorylated in response to 2-deoxyglucose or phenformin, stimuli that activate the AMPK via the upstream kinase LKB1. 2-deoxyglucose stimulated Thr49 phosphorylation of endogenous TPS5 in Arabidopsis cells, whereas phenformin did not. Moreover, extractable SnRK1 activity was increased in Arabidopsis cells in response to 2-deoxyglucose. The plant kinase was inactivated by dephosphorylation and reactivated by phosphorylation with human LKB1, indicating that elements of the SnRK1/AMPK pathway are conserved in Arabidopsis and human cells. We hypothesize that coordinated phosphorylation and 14-3-3 binding of nitrate reductase (NR), 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (F2KP) and class II TPS isoforms mediate responses to signals that activate SnRK1.     

Characterization of the cit-1 gene from Neurospora crassa encoding the mitochondrial form of citrate synthase

We have isolated the cDNA and corresponding genomic DNA encoding citrate synthase in Neurospora crassa. Analysis of the protein coding region of this gene, named cit-1, indicates that it specifies the mitochondrial form of citrate synthase. The predicted protein has 469 amino acids and a molecular mass of 52002 Da. The gene is interrupted by four introns. Hybridization experiments show that a cit-1 probe binds to two different fragments of genomic DNA, which are located on different chromosomes. Neurospora crassa may have two isoforms of citrate synthase, one in the mitochondria and the other in microbodies.     

Characterization of the cit-1 gene from Neurospora crassa encoding the mitochondrial form of citrate synthase

We have isolated the cDNA and corresponding genomic DNA encoding citrate synthase in Neurospora crassa. Analysis of the protein coding region of this gene, named cit-1, indicates that it specifies the mitochondrial form of citrate synthase. The predicted protein has 469 amino acids and a molecular mass of 52002 Da. The gene is interrupted by four introns. Hybridization experiments show that a cit-1 probe binds to two different fragments of genomic DNA, which are located on different chromosomes. Neurospora crassa may have two isoforms of citrate synthase, one in the mitochondria and the other in microbodies.     

Identification of a new isoform of eEF2 whose phosphorylation is required for completion of cell division in sea urchin embryos

Elongation factor 2 (eEF2) is the main regulator of peptide chain elongation in eukaryotic cells. Using sea urchin eggs and early embryos, two isoforms of eEF2 of respectively 80 and 83 kDa apparent molecular weight have been discovered. Both isoforms were identified by immunological analysis as well as mass spectrometry, and appeared to originate from a unique post-translationally modified protein. Accompanying the net increase in protein synthesis that occurs in early development, both eEF2 isoforms underwent dephosphorylation in the 15 min period following fertilization, in accordance with the active role of dephosphorylated eEF2 in regulation of protein synthesis. After initial dephosphorylation, the major 83 kDa isoform remained dephosphorylated while the 80 kDa isoform was progressively re-phosphorylated in a cell-cycle dependent fashion. In vivo inhibition of phosphorylation of the 80 kDa isoform impaired the completion of the first cell cycle of early development implicating the involvement of eEF2 phosphorylation in the exit from mitosis.     

Hypophosphorylated neurofilament subunits undergo axonal transport more rapidly than more extensively phosphorylated subunits in situ

Axonal transport of neurofilaments (NFs) has long been considered to be regulated by phosphorylation. We present evidence that in optic axons of normal mice, the rate of NF axonal transport is inversely correlated with the NF phosphorylation state. In addition to 200 kDa NF-H and 145 kDa NF-M, axonal cytoskeletons from CNS contained a range of phospho-variants of NF-H migrating between 160-200 kDa, and of NF-M migrating at 97-145 kDa. While 160 kDa phospho-variants of NF-H have been well characterized, we confirmed the identity of the previously-described 97 kDa species as a hypophospho-variant of NF-M since (1) pulse-chase metabolic labeling confirmed the 97 kDa species to be a new synthesis product that was converted by phosphorylation over time into a form migrating at 145 kDa, (2) the 97 kDa protein reacted with multiple NF-M antibodies, including one specific for hypophosphorylated NF-M, and (3) dephosphorylation converted NF-M isoforms to 97 kDa. Autoradiographic analyses following metabolic radiolabeling demonstrated that hypophosphorylated NF-H and NF-M isoforms underwent substantially more rapid transport in situ than did extensively phosphorylated isoforms, while NF-H subunits bearing a developmentally delayed C-terminal phospho-epitope transported at a rate slower than that of total 200 kDa NF-H. Differential transport of phospho-variants also highlights that these variants are not homogeneously distributed among NFs, but are segregated to some extent among distinct, although probably overlapping, NF populations, indicating that axonal NFs are not homogeneous with respect to phosphorylation state.     

Signaling Pathways Involved in Dephosphorylation and Localization of the Actin-Binding Protein Cofilin in Stimulated Human Neutrophils

We have studied activation-induced dephosphorylation of proteins in human neutrophils loaded with [³²P]orthophosphate using two-dimensional gel electrophoresis and autoradiography. A major phosphoprotein of 20 kDa in resting neutrophils was markedly dephosphorylated upon activation of cells with chemotactic peptide or phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC). Using a monoclonal anti-cofilin antibody, this phosphoprotein could be shown to be identical with cofilin, a protein implicated in actin filament remodeling. Signaling pathways leading to this dephosphorylation were further characterized. To define the role of PKC isoforms in cofilin dephosphorylation, we used different PKC inhibitors. Gö 6976 (10 μM), which inhibits preferentially PKC α and β, did not prevent PMA-induced dephosphorylation of cofilin, whereas Ro 31-8220 and CGP 41 251 (10 μM), which act also on Ca²⁺-independent PKC isoforms, almost completely suppressed this event. The lack of effect of Gö 6976 was not due to insufficient entry into the cells, as this drug suppressed PMA-induced increases in protein phosphorylation. Ca²⁺-independent PKC isoforms, rather than PKC α or β, may thus be involved in PMA-induced cofilin dephosphorylation. In contrast, Ro 31-8220 did not inhibit chemotactic peptide-induced cofilin dephosphorylation, suggesting here a PKC-independent pathway. The phosphatase inhibitor okadaic acid (1–2 μM) attenuated phosphorylation of cofilin in resting cells. This reduced level was not further attenuated by PMA. Phosphatases 1 and/or 2A may thus control cofilin phosphorylation in resting cells and contribute to PMA-induced cofilin dephosphorylation. Dephosphorylation of cofilin induced by PMA, chemotactic peptide, or okadaic acid was always accompanied by a shift of cofilin to the cell periphery into F-actin-rich areas. These findings suggest a role of cofilin in stimulus-dependent actin remodeling in motile neutrophils.     

Cyclic Nucleotide Dependent Dephosphorylation of Regulator of G-Protein Signaling 18 in Human Platelets

Regulator of G-protein signaling 18 (RGS18) is a GTPase-activating protein that turns off Gq signaling in platelets. RGS18 is regulated by binding to the adaptor protein 14-3-3 via phosphorylated serine residues S49 and S218 on RGS18. In this study we confirm that thrombin, thromboxane A2, or ADP stimulate the interaction of RGS18 and 14-3-3 by increasing the phosphorylation of S49. Cyclic AMP- and cyclic GMP-dependent kinases (PKA, PKG) inhibit the interaction of RGS18 and 14-3-3 by phosphorylating S216. To understand the effect of S216 phosphorylation we studied the phosphorylation kinetics of S49, S216, and S218 using Phos-tag gels and phosphorylation site-specific antibodies in transfected cells and in platelets. Cyclic nucleotide-induced detachment of 14-3-3 from RGS18 coincides initially with double phosphorylation of S216 and S218. This is followed by dephosphorylation of S49 and S218. Dephosphorylation of S49 and S218 might be mediated by protein phosphatase 1 (PP1) which is linked to RGS18 by the regulatory subunit PPP1R9B (spinophilin). We conclude that PKA and PKG induced S216 phosphorylation triggers the dephosphorylation of the 14-3-3 binding sites of RGS18 in platelets.     

In vitro filament formation of a new fiber-forming protein from Tetrahymena pyriformis

Using a 38,000-dalton protein (FFP-38) purified from Tetrahymena acetone powder, we have succeeded in the polymerization of this protein into 14-nm filaments. The polymerization was initiated by incubating the purified FFP-38 fraction in a buffer containing 5 mM Mes (2-(N-Morpholino)ethanesulfonic acid), 50 mM KCl, 1.2 mM CaCl2, 0.6 mM ATP, pH 6.6, and by shifting the incubation temperature from 0 degrees C to 37 degrees C. The 14-nm filament is considered to consist of 7-nm globular subunits regularly arranged into 2 start, helical strands with 4 subunits per turn. The subunit may correspond to 9S tetramer of FFP-38, a native form of FFP-38. Since the subunit arrangement and subunit protein component of this 14-nm filament obviously differ from those of actin filament, 10-nm intermediate filament and microtubule, the 14-nm filament appears to be a newly found intracellular filament. Concerning the FFP-38 polymerization, some polymorphism appeared: we found ring structures having the diameters of 0.3--3.7 micrometers and latticed sheet structure, besides typical straight filaments.     

Balance between activities of Rho kinase and type 1 protein phosphatase modulates turnover of phosphorylation and dynamics of desmin/vimentin filaments

To analyze the cell cycle-dependent desmin phosphorylation by Rho kinase, we developed antibodies specifically recognizing the kinase-dependent phosphorylation of desmin at Thr-16, Thr-75, and Thr-76. With these antibodies, phosphorylation of desmin was observed specifically at the cleavage furrow in late mitotic Saos-2 cells. We then found that treatment of the interphase cells with calyculin A revealed phosphorylation at all the three sites of desmin. We also found that an antibody, which specifically recognizes vimentin phosphorylated at Ser-71 by Rho kinase, became immunoreactive after calyculin A treatment. This calyculin A-induced interphase phosphorylation of vimentin at Ser-71 was blocked by Rho kinase inhibitor or by expression of the dominant-negative Rho kinase. Taken together, our results indicate that Rho kinase is activated not only in mitotic cells but also interphase ones, and phosphorylates intermediate filament proteins, although the apparent phosphorylation level is diminished to an undetectable level due to the constitutive action of type 1 protein phosphatase. The balance between intermediate filament protein phosphorylation by Rho kinase and dephosphorylation by type 1 protein phosphatase may affect the continuous exchange of intermediate filament subunits between a soluble pool and polymerized intermediate filaments.     

Architecture of Ure2p prion filaments: the N-terminal domains form a central core fiber

The [URE3] prion is an inactive, self-propagating, filamentous form of the Ure2 protein, a regulator of nitrogen catabolism in yeast. The N-terminal "prion" domain of Ure2p determines its in vivo prion properties and in vitro amyloid-forming ability. Here we determined the overall structures of Ure2p filaments and related polymers of the prion domain fused to other globular proteins. Protease digestion of 25-nm diameter Ure2p filaments trimmed them to 4-nm filaments, which mass spectrometry showed to be composed of prion domain fragments, primarily residues approximately 1-70. Fusion protein filaments with diameters of 14-25 nm were also reduced to 4-nm filaments by proteolysis. The prion domain transforms from the most to the least protease-sensitive part upon filament formation in each case, implying that it undergoes a conformational change. Intact filaments imaged by cryo-electron microscopy or after vanadate staining by scanning transmission electron microscopy (STEM) revealed a central 4-nm core with attached globular appendages. STEM mass per unit length measurements of unstained filaments yielded 1 monomer per 0.45 nm in each case. These observations strongly support a unifying model whereby subunits in Ure2p filaments, as well as in fusion protein filaments, are connected by interactions between their prion domains, which form a 4-nm amyloid filament backbone, surrounded by the corresponding C-terminal moieties.     

Phosphorylation-dependent interactions between enzymes of plant metabolism and 14-3-3 proteins

Far-Western overlays of soluble extracts of cauliflower revealed many proteins that bound to digoxygenin (DIG)-labelled 14-3-3 proteins. Binding to DIG-14-3-3s was prevented by prior dephosphorylation of the extract proteins or by competition with 14-3-3-binding phosphopeptides, indicating that the 14-3-3 proteins bind to phosphorylated sites. The proteins that bound to the DIG-14-3-3s were also immunoprecipitated from extracts with anti-14-3-3 antibodies, demonstrating that they were bound to endogenous plant 14-3-3 proteins. 14-3-3-binding proteins were purified from cauliflower extracts, in sufficient quantity for amino acid sequence analysis, by affinity chromatography on immobilised 14-3-3 proteins and specific elution with a 14-3-3-binding phosphopeptide. Purified 14-3-3-binding proteins included sucrose–phosphate synthase, trehalose-6-phosphate synthase, glutamine synthetases, a protein (LIM17) that has been implicated in early floral development, an approximately 20 kDa protein whose mRNA is induced by NaCl, and a calcium-dependent protein kinase that was capable of phosphorylating and rendering nitrate reductase (NR) sensitive to inhibition by 14-3-3 proteins. In contrast to the phosphorylated NR-14-3-3 complex which is activated by dissociation with 14-3-3-binding phosphopeptides, the total sugar–phosphate synthase activity in plant extracts was inhibited by up to 40% by a 14-3-3-binding phosphopeptide and the phosphopeptide-inhibited activity was reactivated by adding excess 14-3-3 proteins. Thus, 14-3-3 proteins are implicated in regulating several aspects of primary N and C metabolism. The procedures described here will be valuable for determining how the phosphorylation and 14-3-3-binding status of defined target proteins change in response to extracellular stimuli.     

Bistratene A: a novel compound causing changes in protein phosphorylation patterns in human leukemia cells.

Bistratene A, a polyether toxin isolated from the colonial ascidian Lissoclinum bistratum, causes incomplete differentiation of human leukemia (HL-60) cells apparently through a mechanism not involving protein kinase C. In view of the importance of phosphorylation/dephosphorylation in cellular growth and differentiation we have investigated protein phosphorylation in these cells following exposure to bistratene A, using two-dimensional polyacrylamide gel electrophoresis. Marked increases in the phosphorylation of a protein of 20 kDa, pl 6.7, and a basic protein of 25 kDa were observed after incubation with bistratene A. A comparison was made with cells treated with 12-O-tetradecanoylphorbol 13-acetate and bryostatin 5. While changes in phosphorylation patterns were observed with these two compounds, the 20 kDa and 25 kDa proteins did not undergo phosphorylation changes. The 20 kDa protein was induced rapidly by very low concentrations of bistratene A reaching near maximal levels with 10 nM at 15 min exposure. This protein was found to be localised to the cytoplasm. Phosphoaminoacid analysis demonstrated that the majority of 32P was present in serine and tyrosine residues. The increased phosphorylation of the 20 kDa protein appeared to be due to hyperphosphorylation of existing protein although there was some increase in the amount of the protein. These results suggest that bistratene A will be a useful tool with which to investigate cellular differentiation mechanisms.     

Partial purification and characterization of thrombolamban, a 22,000 dalton cAMP-dependent protein kinase substrate in platelets

In preparations of human platelet microsomes, cyclic AMP-dependent protein kinase induced the rapid phosphorylation of a single protein that was electrophoretically identical to the 22,000 dalton protein (P22) phosphorylated by cAMP in intact platelets. Phosphorylation of the microsomal protein was maximal at one minute and was followed by slow dephosphorylation. Although the protein was associated with a microsomal fraction, it could be separated from the membrane by 2 M NaCl indicating that it was a peripheral protein. Molecular weight was estimated by NaDodSO4-PAGE and by gel filtration chromatography. The molecular weight estimated by NaDodSO4-PAGE was 22,400 daltons and was somewhat larger than the 16,000 molecular weight estimated by gel filtration in the presence of NaDodSO4. In the absence of NaDodSO4, the protein chromatographed as a 36,000 dalton form. The presence of the 36,000 dalton form was not dependent on the phosphorylation state of the protein. The partially purified protein contained phosphoserine, but no phosphothreonine or phosphotyrosine. Two dimensional NaDodSO4-PAGE and isoelectric focusing of the phosphorylated protein revealed isomers with pl values of 5.9 and 6.3. These studies indicate that the 22 kDa microsomal protein and P22 in intact platelets are the same protein and that the 22 kDa protein is tightly bound to the microsomal membrane although the nature of this binding and the microsomal component(s) to which it is bound remain to be determined. We conclude that the 22 kDa protein in platelet microsomes is structurally distinct from, but functionally similar to, phospholamban, the cAMP-dependent protein kinase substrate in muscle, and may play a similar role in calcium transport. Based on this similarity, it is proposed that the 22 kDa protein in platelets be called thrombolamban.     

Role of reversible phosphorylation of acetyl-CoA carboxylase in long-chain fatty acid synthesis

Acetyl-CoA carboxylase, the rate-limiting enzyme in the biogenesis of long-chain fatty acids, is regulated by phosphorylation and dephosphorylation. The major phosphorylation sites that affect carboxylase activity and the specific protein kinases responsible for phosphorylation of different sites have been identified. A form of acetyl-CoA carboxylase that is independent of citrate for activity occurs in vivo. This active form of carboxylase becomes citrate-dependent upon phosphorylation under conditions of reduced lipogenesis. Therefore, phosphorylation-dephosphorylation of acetyl-CoA carboxylase is the enzyme's primary short-term regulatory mechanism; this control mechanism together with cellular metabolites such as CoA, citrate, and palmitoyl-CoA serves to fine-tune the synthesis of long-chain fatty acids under different physiological conditions.     

Characterization of a sperm factor for egg activation at fertilization of the newt Cynops pyrrhogaster

Eggs of the newt, Cynops pyrrhogaster, arrested at the second meiotic metaphase are activated by sperm at fertilization and then complete meiosis to initiate development. We highly purified a sperm factor for egg activation from a sperm extract with several chromatographies. The purified fraction containing only a 45 kDa protein induced egg activation accompanied by an intracellular Ca2+ increase when injected into unfertilized eggs. Although injection of mouse phospholipase C (PLC) zeta-mRNA caused a Ca2+ increase and egg activation, partial amino acid sequences of the 45 kDa protein were homologous to those of Xenopus citrate synthase, but not to PLCs. An anti-porcine citrate synthase antibody recognized the 45 kDa protein both in the purified fraction and in the sperm extract. Treatment with the anti-citrate synthase antibody reduced the egg-activation activity in the sperm extract. Injection of porcine citrate synthase or mRNA of Xenopus citrate synthase induced a Ca2+ increase and caused egg activation. A large amount of the 45 kDa protein was localized in two lines elongated from the neck to the middle piece of sperm. These results indicate that the 45 kDa protein is a major component of the sperm factor for egg activation at newt fertilization.     

Syncoilin isoform organization and differential expression in murine striated muscle

Syncoilin is a 64 kDa intermediate filament (IF) protein expressed in myocytes at the sarcolemma, perinucleus, myotendenous and neuromuscular junctions. Here we present a revised domain projection and structural analysis for the original isoform (sync-1) and introduce two novel syncoilin isoforms (sync-2 and sync-3) generated by exon splicing. On the basis of consensus identity we propose that syncoilin be reclassified as a type III IF protein. All three syncoilin isoforms lack a L1 domain, a significant departure from standard IF rod domain projections that is likely to impact significantly on their biological function. Our analyses indicate that syncoilin is unlikely to form classical intermediate filament structures by itself, and that the significant difference in C-terminal structure between the three isoforms indicates that they may play divergent roles in myocytes. We show that despite lacking an apparent structural role in striated muscle, syncoilin isoforms are differentially and strongly upregulated in response to cardiotoxin induced regeneration and denervation induced atrophy in the C57BL/6 mouse, possibly suggesting an atypical role for syncoilin in muscle.     

Platelet talin is phosphorylated by calyculin A

Calyculin A and okadaic acid, potent and cell permeable inhibitors of type 1 and type 2A protein phosphatases, inhibit platelet aggregation and secretion. However, the relationship between phosphatase inhibition and inhibition of platelet function is not well understood. We found that in unstimulated platelets, talin (P235) was phosphorylated at threonine residues by calyculin A. Furthermore, the extent of talin phosphorylation by calyculin A was closely correlated with its inhibition of thrombin-induced platelet aggregation. Since the binding of talin to platelet glycoprotein IIb/IIIa complex has been shown to be affected by its phosphorylation, these results suggest that type 1 and/or type 2A protein phosphatases may play a role in the regulation of membrane-cytoskeleton interaction through dephosphorylation of talin.     

The effects of platelet secretion inhibitors on protein phosphorylation

Protein phosphorylation was investigated in human platelets after stimulation to secretion by thrombin. After stimulation by thrombin at 4 degrees C (in which secretion is inhibited), phosphorylations of the 80, 56, and 38 kDa polypeptides and dephosphorylation of the 67 kDa phosphopeptide eventually occurred. The phosphorylations of the 27 and 20 kDa polypeptides remained inhibited until the temperature was increased to 37 degree C, which also resulted in secretion. Various stimulants and inhibitors of platelet function were used to characterize individual protein phosphorylations. The divalent-cation ionophore, A23187, induced the phosphorylations (or dephosphorylation) of the same proteins as thrombin with the exception of the 80 kDa protein, which remained incompletely phosphorylated. The intracellular calcium antagonist, TMB-8, inhibited thrombin-stimulated secretion and phosphorylation of all the polypeptides except the 80 kDa protein. The dephosphorylation of the 67 kDa phosphoprotein was not affected by TMB-8. Incubation of platelets with prostaglandin E1 and isobutylmethylxanthine inhibited thrombin-stimulated secretion and the phosphorylation of the 38 and 20 kDa protein and increased the phosphorylation of the 67 and 27 kDa phosphoproteins. These observations may be used to correlate protein phosphorylation with secretion, suggesting a possible sequence of intracellular events that mediate thrombin-stimulated secretion.