Cell surface phosphorylation by a novel ecto-protein kinase: a key regulator of cellular functions in spermatozoa

Since 1976 many studies have been reported on the occurrence and functional significance of ecto-protein kinases in a variety of cell types although their precise biochemical identity is largely unknown. This study reports for the first time purification to apparent homogeneity of an ecto-protein kinase (ecto-CIK) and some of its characteristics using caprine sperm as the cell model. The ecto-CIK is a unique membrane-specific serine/threonine protein kinase. It is a strongly basic 115 kDa protein made up of two subunits: 63 and 55 kDa. The ecto-kinase undergoes a remarkable lateral movement on the outer cell surface culminating in capping on the sperm acrosomal tip. MPS, its major protein substrate is also located on the acrosomal tip. Both ecto CIK and MPS serve as potential regulators of flagellar motility. This novel enzyme appears to be major kinase responsible for the reported regulation of mammalian cellular functions by modulating phosphorylation of the membrane-bound proteins.     

A novel, carbohydrate signal-mediated cell surface protein phosphorylation: ganglioside GQ1b stimulates ecto-protein kinase activity on the cell surface of a human neuroblastoma cell line, GOTO

A ganglioside-stimulated ecto-type protein phosphorylation system (ecto-Gg-kinase) was detected on the cell surface of a human neuroblastoma cell line (GOTO). When intact cells were incubated with [gamma-32P]ATP, at least 28 cell surface proteins were phosphorylated, as evident on SDS-PAGE (4-20%) analysis. Exogenously added gangliosides specifically stimulated the phosphorylation of at least three cell surface associated proteins of Mr = 64,000, 60,000, and 54,000. Phosphorylation was directed toward Thr and Ser residues, respectively, as revealed on acid hydrolysis followed by electrophoresis. GQ1b, at 5 nM, was the most potent among the several gangliosides tested and was more effective when added to cells before [gamma-32P]ATP administration. The simultaneous addition of an excess amount of the saccharide portion of GQ1b (oligo-GQ1b) inhibited the GQ1b-stimulated phosphorylation, indicating the necessity of the sialosaccharide moiety. These results strongly suggest that phosphorylation of the three proteins may be closely associated with the highly specific neuritogenic effect of GQ1b previously reported.     

Phosphorylation of external cell-surface proteins by an endogenous ecto-protein kinase of goat epididymal intact spermatozoa

Intact spermatozoa from goat cauda epididymides possess an ecto-(cyclic AMP-independent protein kinase) activity that causes transfer of the terminal phosphate of exogenously added [gamma-32P]ATP to the serine and threonine residues of several endogenous plasma-membrane phosphoproteins located on the external cell surface. Cyclic AMP, cyclic GMP, calmodulin and muscle cyclic AMP-dependent protein kinases I and II had no appreciable effect on the rate of phosphorylation of ecto-proteins by the intact cells. The ecto-enzyme is not derived from the catalytic subunit of a cyclic AMP-dependent kinase. Sperm ecto-kinase activity is not due to contamination of broken cells or any possible cell damage during incubation and isolation of spermatozoa. The phosphorylation reaction was linear for approx. 1 min and there was no detectable uptake of ATP by these cells. The activity of the ecto-kinase was strongly inhibited by proteinases and by the membrane-nonpenetrating surface probes. The products of the reaction were associated with the intact cells and the 32P of the labelled cells was largely lost when treated with Triton X-100 or proteinases: trypsin and pronase. These data are consistent with the view that the observed protein kinase and the phosphoproteins are located on the external surface of spermatozoa. Vigorously forward-motile whole spermatozoa showed a relatively high capacity to phosphorylate ecto-proteins that undergo rapid turnover. The results suggest the occurrence of a novel coupled-enzyme system (ecto-protein kinase and phosphoprotein phosphatase) on the sperm external surface that may modulate sperm physiology by determining the phosphorylated states of the ecto-proteins.     

Activation of a cellular tyrosine-specific protein kinase by phosphorylation

One of the 3 major RNA-binding proteins of rabbit reticulocytes, a polypeptide of 36 kDa, is identified as glyceraldehyde-3-phosphate dehydrogenase (GAPD). This fact was deduced from the identity of molecular masses, one-dimensional peptide maps and isoelectric points of the 36 kDa protein and GAPD from rabbit muscle. It is concluded that GAPD can bind rather unspecifically different RNAs and polynucleotides. This means that GAPD, like other RNA-binding proteins, can form loose dynamic complexes with polyribosomes. Association of such a kind may be used for compartmentation of glycolysis near polyribosomes.     

Sphingosine kinase: a mediator of vital cellular functions

Ample evidence indicates that sphingosine-1-phosphate (SPP) can serve as an intracellular second messenger regulating calcium mobilization, and cell growth and survival. Moreover, the dynamic balance between levels of the sphingolipids metabolites, ceramide and SPP, and consequent regulation of opposing signaling pathways, is an important factor that determines whether a cell survives or dies. SPP has also recently been shown to be the ligand for the EDG-1 family of G protein-coupled receptors, which now includes EDG-1, -3, -5, -6, and -8. SPP is thus a lipid mediator that has novel dual actions signaling inside and outside of the cell. This review is focussed on sphingosine kinase, the enzyme that regulates levels of SPP and thus plays a critical role in diverse biological processes.     

Sphingosine kinase: a mediator of vital cellular functions

Ample evidence indicates that sphingosine-1-phosphate (SPP) can serve as an intracellular second messenger regulating calcium mobilization, and cell growth and survival. Moreover, the dynamic balance between levels of the sphingolipids metabolites, ceramide and SPP, and consequent regulation of opposing signaling pathways, is an important factor that determines whether a cell survives or dies. SPP has also recently been shown to be the ligand for the EDG-1 family of G protein-coupled receptors, which now includes EDG-1, -3, -5, -6, and -8. SPP is thus a lipid mediator that has novel dual actions signaling inside and outside of the cell. This review is focussed on sphingosine kinase, the enzyme that regulates levels of SPP and thus plays a critical role in diverse biological processes.     

Protein kinase C zeta. A novel regulator of both phosphorylation and de-phosphorylation of cardiac sarcomeric proteins

Our experiments investigated associations of specific isoforms of protein kinase C (PKC) with individual proteins in the cardiac troponin complex. Troponin I (cTnI) associated with PKCepsilon and zeta and troponin T (cTnT) associated with PKC alpha, delta, and epsilon. Based on its association with cTnI, we hypothesized that PKCzeta is a major regulator of myofilament protein phosphorylation. To test this, we infected adult cardiac myocytes with adenoviral constructs containing DsRed monomer-tagged wild type (WT) and the following constitutively active forms of PKCzeta: the pseudo-substrate region (A119E), 3'-phospho-inositide-dependent kinase-1 (T410E), and auto-phosphorylation (T560E). The A119E and T410E mutants displayed increased localization to the Z-discs compared with WT and T560E. Immunoprecipitations were performed in myocytes expressing PKCzeta using PKC phospho-motif antibodies to determine the phosphorylation of cTnI, cTnT, tropomyosin, myosin-binding protein C, and desmin. We did not find serine (Ser) phosphorylation of cTnI or cTnT. However, we observed a significant decrease in threonine (Thr) phosphorylation of cTnI and cTnT notably by PKCzeta T560E. Ser phosphorylation of tropomyosin was increased by all three active mutants of PKCzeta. Ser/Thr phosphorylation of myosin-binding protein C increased primarily by PKCzeta A119E. Both PKCzeta A119E and T410E mutants increased desmin Ser/Thr phosphorylation. To explain the apparent Thr dephosphorylation of cTnI and cTnT, we hypothesized that PKCzeta exists as a complex with p21-activated kinase-1 (Pak1) and protein phosphatase 2A (PP2A), and this was confirmed by immunoprecipitation Western blot. Our data demonstrate that PKCzeta is a novel regulator of myofilament protein phosphorylation.     

RasGAPs: a crucial regulator of extracellular stimuli for homeostasis of cellular functions

Ras and its GTPase activating proteins (GAPs) are among the crucial regulators of extracelluar ligands. Information about these regulators has been elucidated during the course of studies in signal transduction over the last two decades. RasGAPs such as p120GAP and neurofibromin have been studied extensively for their roles as either "negative" regulators or effectors of Ras. Accumulating evidence suggests that these molecules are crucial regulators of extracellular stimuli that serve to maintain the homeostasis of cellular functions. This compendium highlights cellular functions of RasGAPs and their signaling characteristics from the viewpoint of homeostasis, including our recent finding of the phenotype of R-RasGAP mutant mice whose GAP activity is down-regulated.     

Phosphorylation and dephosphorylation of human platelet surface proteins by an ecto-protein kinase/phosphatase system.

We have characterized a novel ecto-protein kinase activity and a novel ecto-protein phosphatase activity on the membrane surface of human platelets. Washed intact platelets, when incubated with [gamma-32P]ATP in Tyrode's buffer, showed the phosphorylation of a membrane surface protein migrating with an apparent molecular mass of 42 kDa on 5-15% SDS polyacrylamide gradient gels. The 42 kDa protein could be further resolved on 15% SDS gels into two proteins of 39 kDa and 42 kDa. In this gel system, it was found that the 39 kDa protein became rapidly phosphorylated and dephosphorylated, whereas the 42 kDa protein was phosphorylated and dephosphorylated at a much slower rate. NaF inhibited the dephosphorylation of these proteins indicating the involvement of an ecto-protein phosphatase. The platelet membrane ecto-protein kinase responsible for the phosphorylation of both of these proteins was identified as a serine kinase and showed dependency on divalent cations Mg2+ or Mn2+ ions. Ca2+ ions potentiated the Mg(2+)-dependent ecto-protein kinase activity. The ecto-protein kinase rapidly phosphorylated histone and casein added exogenously to the extracellular medium of intact platelets. Following activation of platelets by alpha-thrombin, the incorporation of [32P]phosphate from exogenously added [gamma-32P]ATP by endogenous protein substrates was reduced by 90%, suggesting a role of the ecto-protein kinase system in the regulation of platelet function. The results presented here demonstrate that both protein kinase and protein phosphatase activities reside on the membrane surface of human platelets. These activities are capable of rapidly phosphorylating and dephosphorylating specific surface platelet membrane proteins which may play important roles in early events of platelet activation and secretion.     

A tobacco calcium/calmodulin-binding protein kinase functions as a negative regulator of flowering

A tobacco calcium/calmodulin-binding protein kinase (NtCBK1) was isolated and identified. The predicted NtCBK1 protein has 599 amino acids, an N-terminal kinase domain, and shares high homology with other calmodulin (CaM)-related kinases. Whereas NtCBK1 phosphorylates itself and substrates such as histone IIIS and syntide-2 in the absence of CaM, its kinase activity can be stimulated by tobacco CaMs. However, unlike another tobacco protein kinase designated NtCBK2, NtCBK1 was not differentially regulated by the different CaM isoforms tested. The CaM-binding domain of NtCBK1 was located between amino acids 436 and 455, and this domain was shown to be necessary for CaM modulation of kinase activity. RNA in situ hybridization showed that NtCBK1 was highly regulated in the transition to flowering. Whereas NtCBK1 mRNA was accumulated in the shoot apical meristem during vegetative growth, its expression was dramatically decreased in the shoot apical meristem after floral determination, and in young flower primordia. The expression of NtCBK1 was up-regulated to high levels in floral organ primordia. Fluctuations in NtCBK1 expression were verified by analysis of tobacco plants expressing green fluorescent protein under the control of the NtCBK1 promoter, suggesting a role of NtCBK1 in the transition to flowering. This conclusion was confirmed by overexpressing NtCBK1 in transgenic tobacco plants, where maintenance of high levels of NtCBK1 in the shoot apical meristem delayed the switch to flowering and extended the vegetative phase of growth. Further work indicated that overexpression of NtCBK1 in transgenic tobacco did not affect the expression of NFL, a tobacco homologue of the LFY gene that controls meristem initiation and floral structure in tobacco. In addition, the promotion of tobacco flowering time by DNA demethylation cannot be blocked by the overexpression of NtCBK1.     

Focal adhesion kinase: protein interactions and cellular functions

Integrin-mediated cell adhesion to extracellular matrix (ECM) plays important roles in a variety of biological processes. Recent studies suggested that integrins mediate signal transduction across the plasma membrane via activating several intracellular signaling pathways. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that has been shown to be a major mediator of integrin signal transduction pathways. Upon activation by integrins, FAK undergoes autophosphorylation as well as associations with several other intracellular signaling molecules. These interactions in the signaling pathways have been shown to regulation a variety of cellular functions such as cell spreading, migration, cell proliferation, apoptosis and cell survival. Recent progress in the understanding of FAK interactions with other proteins in the regulation of these cellular functions will be discussed in this review.     

A novel fold revealed by Mycobacterium tuberculosis NAD kinase, a key allosteric enzyme in NADP biosynthesis

NAD kinase catalyzes the magnesium-dependent phosphorylation of NAD, representing the sole source of freshly synthesized NADP in all organisms. The enzyme is essential for the growth of the deadly multidrug-resistant pathogen Mycobacterium tuberculosis and is an attractive target for novel antitubercular agents. The crystal structure of NAD kinase has been solved by multiwavelength anomalous dispersion at a resolution of 2.3 A in its T state. Two crystal forms have been obtained revealing either a dimer or a tetramer. The enzyme architecture discloses a novel molecular arrangement, with each subunit consisting of an alpha/beta N-terminal domain and a C-terminal 12-stranded beta sandwich domain, connected by swapped beta strands. The C-terminal domain shows a striking internal approximate 222 symmetry and an unprecedented topology, revealing a novel fold within the family of all beta structures. The catalytic site is located in the long crevice that defines the interface between the domains. The conserved GGDG structural fingerprint of the catalytic site is reminiscent of the related region in 6-phosphofructokinase, supporting the hypothesis that NAD kinase belongs to a newly reported superfamily of kinases.     

Fascin: A key regulator of cytoskeletal dynamics

Fascin is a 55 kDa actin-bundling protein and is an important regulatory element in the maintenance and stability of parallel bundles of filamentous actin in a variety of cellular contexts. Regulation of fascin function is under the control of a number of different signalling pathways that act in concert to spatially regulate the actin-binding properties of this protein. The ability of fascin to bind and bundle actin plays a central role in the regulation of cell adhesion, migration and invasion. Fascin has received considerable attention recently as an emerging key prognostic marker of metastatic disease. Studies are now underway to better understand the precise regulation of this protein in the context of tumour progression and to investigate fascin as a potential therapeutic target for a number of forms of cancer.     

Cell cycle arrest and cellular differentiation mediated by a cell surface sialoglycopeptide

Cell cycling by a relatively wide variety of cell lines was shown to be reversibly inhibited by a cell surface sialoglycopeptide (SGP) isolated and purified from intact bovine cerebral cortex cells. Cell cycle arrest, mediated by the bovine SGP inhibitor, was shown to be completely reversible with mouse Swiss 3T3, mouse 1316 fibrosarcoma, mouse N2a neuroblastoma, bovine MDBK and monkey BSC-1 cells. These cell lines represented both fibroblast and epithelial-like cells, transformed and nontransformed cells, as well as their being derived from a broad array of species. In contrast to the others tested, human HL-60 leukemic cells were sensitive to the inhibitory effects of the SGP but did not reenter the mitotic cycle after the removal of the inhibitor. Instead, the mitotic arrest of HL-60 cells appeared to enhance entry into a terminal and irreversible state of cellular differentiation.