Ca2+/calmodulin-dependent protein kinase V and I may form a family of isoforms

We reported that polyclonal antibody against Ca2+/calmodulin-dependent protein kinase V (CaM kinase V) reacted to two proteins of rat cerebrum with a molecular mass of 40 and 41 kDa. This antibody revealed the immunoreactivity with CaM kinase I expressed in E. coli (recombinant CaM kinase I), of which molecular mass was 40 kDa, whereas 41 kDa mainly with purified CaM kinase V. The immunoreactive bands of recombinant CaM kinase I and CaM kinase V did not shift by phosphorylation or dephosphorylation. These results suggest that CaM kinase V and CaM kinase I may form a family of isoforms.     

Follistatin: a multifunctional regulatory protein

Follistatin was first described in 1987 as a follicle-stimulating hormone inhibiting substance present in ovarian follicular fluid. We now know that this effect of follistatin is only one of its many properties in a number of reproductive and nonreproductive systems. A majority of these functions are facilitated through the affinity of follistatin for activin, where activin's effects are neutralized through its binding to follistatin. As such, the interplay between follistatin and activin represents a powerful regulatory mechanism that impinges on a variety of cellular processes within the body. In this review we focus on the biochemical characteristics of follistatin and its interaction with activin and discuss the emerging role of these proteins as potent tissue regulators in the gonad, pituitary gland, pregnancy membranes, vasculature, and liver. Consideration is also given to the larger family of proteins that contain follistatin-like modules, in particular with regard to their functional and structural implications.     

Casein kinase I alpha and alpha L: alternative splicing-generated kinases exhibit different catalytic properties

Casein kinase I (CKI) is a family of serine/threonine protein kinases found in all eukaryotes examined to date. Here, the rat CKI isoforms alpha and alpha L were cloned and expressed in both eukaryotic and prokaryotic systems. Characterization of the genomic DNA flanking the exon unique to CKI alpha L demonstrated that CKI alpha and CKI alpha L arise by the alternative splicing of a common pre-mRNA molecule. To the best of our knowledge, the alpha L isoform is the only known active serine/threonine kinase to contain an insert within its catalytic domain. Tissue distribution of each splicing isoform was examined by RT-PCR, immunoprecipitation, and Western blotting. Both isoforms were expressed in all tissues tested but at different levels. Bacterially expressed CKI alpha isoforms were active and therefore biochemically characterized. CKI alpha and CKI alpha L proteins were demonstrated to have casein kinase I catalytic properties. More importantly, the recombinant isoform proteins exhibited differences in binding and activity toward common CKI substrates. These observations demonstrate that the alpha L insert within the kinase domain modulates substrate kinetics. These kinetic differences suggest that CKI alpha and CKI alpha L may perform different biological roles.     

New isoforms of multifunctional calcium/calmodulin-dependent protein kinase II

Calcium/calmodulin dependent protein kinase II (CaM kinase II) seems to act as an important regulator of intracellular signal transmission. Four subtypes, termed alpha to delta, have been cloned; some of them can exist as different splicing variants. All these isoforms share a great overall homology, and they contain 3 areas of low homology. We have identified 5 new variants of subtype delta so that the total number of different isoforms now adds up to 12. These variants are probably a result of different splicing and show several deletions in regard to subtype delta. The deletion sites do exactly match regions of low homology between the subtypes. This suggests a functional division of the CaM kinase II molecule into homologous and variable domains. The homologous domains are highly conserved. Therefore, it might be the case that the constitution of the variable domains is more significant for a certain isoform than its belonging to one of the 4 subtypes alpha to delta.     

Casein kinase II stimulates Xenopus laevis DNA topoisomerase I by physical association

A Xenopus laevis casein kinase II-like activity copurified with X. laevis DNA topoisomerase I activity during chromatography on DEAE-cellulose, phosphocellulose, and hydroxylapatite, but the two activities were resolved by chromatography on DNA-agarose [Kaiserman, H. B., Ingebritsen, T. S., & Benbow, R. M. (1988) Biochemistry 27, 3216-3222]. Phosphorylation of the catalytic polypeptides of dephosphorylated X. laevis DNA topoisomerase I by the endogenous X. laevis casein kinase II-like activity apparently resulted in a severalfold increase in catalytic activity. In this study, we show that incubation of purified X. laevis DNA topoisomerase I with electrophoretically homogeneous bovine brain casein kinase II and ATP strongly stimulated catalytic activity. Surprisingly, purified bovine casein kinase II stimulated X. laevis DNA topoisomerase I activity by more than an order of magnitude in the absence of ATP, although ATP resulted in additional stimulation. Other basic proteins, such as histone H1 and HMG proteins, also stimulated X. laevis DNA topoisomerase I catalytic activity 2-3-fold in the absence of ATP. Modulation of catalytic activity by direct physical association (protein-protein interactions) must, therefore, be considered in addition to phosphorylation in assessing the physiological role of casein kinase II and other basic proteins during regulation of X. laevis DNA topoisomerase I activity in vivo.     

Purification and characterization of a novel protein activator of Ca2+/calmodulin-dependent protein kinase I

A protein activator of Ca2+/calmodulin (CaM)-dependent protein kinase I was purified from rat brain. The activator was retained on a CaM-Sepharose column in the presence of Ca2+ and kinase assay of renatured gel revealed the 64 kDa molecule in the purified activator fraction to be autophosphorylated and to phosphorylate recombinant CaM kinase I in the presence of Ca2+/calmodulin. These results suggest that this activator of CaM kinase I is also a CaM-dependent protein kinase. Phosphorylation of CaM kinase I by the activator resulted in drastic potentiation of its CaM-dependent activity. Furthermore, kinetic analyses demonstrated that the activation decreases the Km values of CaM kinase I for both ATP and syntide-2 without a change in Vmax values. Considering the quite low enzymatic activity of recombinant CaM kinase I without activation, the 64 kDa species might be essential for CaM kinase I function in vivo.     

Tyrosine phosphorylation-dependent and -independent associations of protein kinase C-delta with Src family kinases in the RBL-2H3 mast cell line: regulation of Src family kinase activity by protein kinase C-delta

Src kinases and protein kinase C (PKC) have been well studied for their role in oncogenic and normal cellular processes. Herein we report on a novel regulatory pathway mediated by the interaction of PKC-delta with p53/56Lsy (Lyn) and with p60Src (Src) that results in the phosphorylation and increased activity of Lyn and Src. In the RBL-2H3 mast cell line, the interaction of PKC-delta with Lyn required the activation of the high affinity receptor for IgE (FcsigmaRI) while the interaction with Src was constitutive. Increased complex formation of PKC-delta with Lyn or Src led to increased serine phosphorylation and activity of the Src family kinases. Conversely, Lyn was found to phosphorylate Lyn-associated and recombinant PKC-delta in vitro and the tyrosine 52 phosphorylated PKC-delta was recruited to associate with the Lyn SH2 domain. The constitutive association of PKC-delta with Src did not result in the tyrosine phosphorylation of PKC-delta prior to or after FsigmaRI engagement. However in cells over-expressing PKC-delta, FsigmaRI engagement resulted in the dramatic inhibition of Src activity and some inhibition of Lyn activity. Thus, the interaction and cross-talk of PKC-delta with Src family kinases suggests a novel and inter-dependent mechanism for regulation of enzymatic activity that may serve an important role in cellular responses.     

Actin cytoskeleton organization regulated by the PAK family of protein kinases

Cdc42, Rac1 and other Rho-type GTPases regulate gene expression, cell proliferation and cytoskeletal architecture [1,2]. A challenge is to identify the effectors of Cdc42 and Rac1 that mediate these biological responses. Protein kinases of the p21-activated kinase (PAK) family bind activated Rac1 and Cdc42, and switch on mitogen-activated protein (MAP) kinase pathways; however, their roles in regulating actin cytoskeleton organization have not been clearly established [3-5]. Here, we show that mutants of the budding yeast Saccharomyces cerevisiae lacking the PAK homologs Ste20 and Cla4 exhibit actin cytoskeletal defects, in vivo and in vitro, that resemble those of cdc42-1 mutants. Moreover, STE20 overexpression suppresses cdc42-1 growth defects and cytoskeletal defects in vivo, and Ste20 kinase corrects the actin-assembly defects of permeabilized cdc42-1 cells in vitro. Thus, PAKs are effectors of Cdc42 in pathways that regulate the organization of the cortical actin cytoskeleton.     

Reconstructing a large-scale spatial arrangement: Effects of environmental organization and operativity.

The effect of the organization of items in a room-sized space on memory for an environmental layout was examined in relation to operative level in classification. 130 5–9 yr olds were pretested and categorized as high, transitional, or low in operativity. Subsequently, Ss were asked to reconstruct 1 of 4 arrangements of 16 items of furniture. The 4 conditions varied in degree of logical organization, clustering of items by similar physical attributes, and meaningfulness of spatial orientation of items. Clustered and meaningful arrangements were expected to facilitate memory, and operativity was expected to further enhance the effects of each. Reconstructions were scored for the accuracy of the specific placement of items, the general placement of items, and spatial relations between items. Clustering increased memory for spatial relations and for the specific and general area placement of items. Meaningful orientation of items in the layout interacted with clustering to improve memory for the general area of the room in which items belonged. Operative knowledge of classification increased memory for general location but did not show the expected interactions with environmental organization. The impact of environmental organization, knowledge of classification, and other age-related changes for memory of large-scale arrangement are discussed. (19 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Activation of a calcium-calmodulin-dependent protein kinase I cascade in PC12 cells

It has been observed that the activity of Ca2+-calmodulin (CaM)-dependent protein kinase I is enhanced up to 50-fold by its phosphorylation in vitro by a distinct CaM kinase I kinase (Lee, J. C., and Edelman, A. M. (1994) J. Biol. Chem. 269, 2158-2164). It has, however, been unclear whether this event represents an acute form of cellular regulation. We demonstrate here the phosphorylation and activation of CaM kinase I in PC12 pheochromocytoma cells in response to elevation of intracellular Ca2+. Treatment of PC12 cells with the Ca2+-ionophore, ionomycin, or with a depolarizing concentration of KCl, led to rapid, biphasic phosphorylation of CaM kinase I and to increases in CaM kinase I activity of 5.1- and 7. 3-fold, respectively. Depolarization-induced activation of CaM kinase I was reduced by approximately 80% by blockade of Ca2+ influx through L-type voltage-dependent Ca2+ channels and completely abolished by removal of extracellular Ca2+. The ability of PC12 cell CaM kinase I to be phosphorylated and activated by purified CaM kinase I kinase in vitro was markedly reduced by prior depolarization of the cells, consistent with intracellular phosphorylation and activation of CaM kinase I by CaM kinase I kinase. These results demonstrate the existence in PC12 cells of a CaM kinase I cascade, the function of which may be to sensitize cells to signal-induced elevations of intracellular Ca2+.     

Three-dimensional structure of human protein kinase C interacting protein 1, a member of the HIT family of proteins

The three-dimensional structure of protein kinase C interacting protein 1 (PKCI-1) has been solved to high resolution by x-ray crystallography using single isomorphous replacement with anomalous scattering. The gene encoding human PKCI-1 was cloned from a cDNA library by using a partial sequence obtained from interactions identified in the yeast two-hybrid system between PKCI-1 and the regulatory domain of protein kinase C-beta. The PKCI-1 protein was expressed in Pichia pastoris as a dimer of two 13.7-kDa polypeptides. PKCI-1 is a member of the HIT family of proteins, shown by sequence identity to be conserved in a broad range of organisms including mycoplasma, plants, and humans. Despite the ubiquity of this protein sequence in nature, no distinct function has been shown for the protein product in vitro or in vivo. The PKCI-1 protomer has an alpha+beta meander fold containing a five-stranded antiparallel sheet and two helices. Two protomers come together to form a 10-stranded antiparallel sheet with extensive contacts between a helix and carboxy terminal amino acids of a protomer with the corresponding amino acids in the other protomer. PKCI-1 has been shown to interact specifically with zinc. The three-dimensional structure has been solved in the presence and absence of zinc and in two crystal forms. The structure of human PKCI-1 provides a model of this family of proteins which suggests a stable fold conserved throughout nature.     

Identification of a novel protein kinase A anchoring protein that binds both type I and type II regulatory subunits

Compartmentalization of cAMP-dependent protein kinase is achieved in part by interaction with A-kinase anchoring proteins (AKAPs). All of the anchoring proteins identified previously target the kinase by tethering the type II regulatory subunit. Here we report the cloning and characterization of a novel anchoring protein, D-AKAP1, that interacts with the N terminus of both type I and type II regulatory subunits. A novel cDNA encoding a 125-amino acid fragment of D-AKAP1 was isolated from a two-hybrid screen and shown to interact specifically with the type I regulatory subunit. Although a single message of 3.8 kilobase pairs was detected for D-AKAP1 in all embryonic stages and in most adult tissues, cDNA cloning revealed the possibility of at least four splice variants. All four isoforms contain a core of 526 amino acids, which includes the R binding fragment, and may be expressed in a tissue-specific manner. This core sequence was homologous to S-AKAP84, including a mitochondrial signal sequence near the amino terminus (Lin, R. Y., Moss, S. B., and Rubin, C. S. (1995) J. Biol. Chem. 270, 27804-27811). D-AKAP1 and the type I regulatory subunit appeared to have overlapping expression patterns in muscle and olfactory epithelium by in situ hybridization. These results raise a novel possibility that the type I regulatory subunit may be anchored via anchoring proteins.     

MKBP, a novel member of the small heat shock protein family, binds and activates the myotonic dystrophy protein kinase

Muscle cells are frequently subjected to severe conditions caused by heat, oxidative, and mechanical stresses. The small heat shock proteins (sHSPs) such as alphaB-crystallin and HSP27, which are highly expressed in muscle cells, have been suggested to play roles in maintaining myofibrillar integrity against such stresses. Here, we identified a novel member of the sHSP family that associates specifically with myotonic dystrophy protein kinase (DMPK). This DMPK-binding protein, MKBP, shows a unique nature compared with other known sHSPs: (a) In muscle cytosol, MKBP exists as an oligomeric complex separate from the complex formed by alphaB-crystallin and HSP27. (b) The expression of MKBP is not induced by heat shock, although it shows the characteristic early response of redistribution to the insoluble fraction like other sHSPs. Immunohistochemical analysis of skeletal muscle cells shows that MKBP localizes to the cross sections of individual myofibrils at the Z-membrane as well as the neuromuscular junction, where DMPK has been suggested to be concentrated. In vitro, MKBP enhances the kinase activity of DMPK and protects it from heat-induced inactivation. These results suggest that MKBP constitutes a novel stress-responsive system independent of other known sHSPs in muscle cells and that DMPK may be involved in this system by being activated by MKBP. Importantly, since the amount of MKBP protein, but not that of other sHSP family member proteins, is selectively upregulated in skeletal muscle from DM patients, an interaction between DMPK and MKBP may be involved in the pathogenesis of DM.     

Physiological phosphorylation of protein kinase A at Thr-197 is by a protein kinase A kinase

Phosphorylation of the catalytic subunit of cyclic AMP-dependent protein kinase, or protein kinase A, on Thr-197 is required for optimal enzyme activity, and enzyme isolated from either animal sources or bacterial expression strains is found phosphorylated at this site. Autophosphorylation of Thr-197 occurs in Escherichia coli and in vitro but is an inefficient intermolecular reaction catalyzed primarily by active, previously phosphorylated molecules. In contrast, the Thr-197 phosphorylation of newly synthesized protein kinase A in intact S49 mouse lymphoma cells is both efficient and insensitive to activators or inhibitors of intracellular protein kinase A. Using [35S]methionine-labeled, nonphosphorylated, recombinant catalytic subunit as the substrate in a gel mobility shift assay, we have identified an activity in extracts of protein kinase A-deficient S49 cells that phosphorylates catalytic subunit on Thr-197. The protein kinase A kinase activity partially purified by anion-exchange and hydroxylapatite chromatography is an efficient catalyst of protein kinase A phosphorylation in terms of both a low Km for ATP and a rapid time course. Phosphorylation of wild-type catalytic subunit by the kinase kinase activates the subunit for binding to a pseudosubstrate peptide inhibitor of protein kinase A. By both the gel shift assay and a [gamma-32P]ATP incorporation assay, the enzyme is active on wild-type catalytic subunit and on an inactive mutant with Met substituted for Lys-72 but inactive on a mutant with Ala substituted for Thr-197. Combined with the results from mutant subunits, phosphoamino acid analysis suggests that the enzyme is specific for phosphorylation of Thr-197.     

Imagining projective transformations: aligned orientations in spatial organization

Four experiments were conducted to investigate whether variations in orientation that profoundly affect the ability to imagine rotations also affect the ability to imagine projective transformations. For a basic rectilinear object and the three simpler Platonic Solids, imagining projective transformations (e.g., the casting of a shadow) was quite successful when the objects were aligned with the direction of projection. For the solids, this alignment occurred when the objects were generalized cylinders about axes aligned with the projection. As the objects were made more oblique to the projection, performance deteriorated markedly. When the objects were moderately aligned with the projection, performance depended on the orientation of the object and the orientation of the projection to the environment. We suggest that the imagination of projection and of rotation is a type of problem solving in which spatial structures are organized in relation to initially given properties of the objects and transformations. When there is alignment among the various structural components, this process of imagination works efficiently. Without such alignment, nonexperts often fail. We suggest that aligned (i.e., parallel and perpendicular) orientations are effective in spatial imagination because they are categorically distinct and singular, and they provide a critical form of redundancy.     

Precise spatial positioning of chromosomes during prometaphase: evidence for chromosomal order

The relative locations of several chromosomes within wheel-shaped prometaphase chromosome rosettes of human fibroblasts and HeLa cells were determined with fluorescence hybridization. Homologs were consistently positioned on opposite sides of the rosette, which suggests that chromosomes are separated into two haploid sets, each derived from one parent. The relative locations of chromosomes on the rosette were mapped by dual hybridizations. The data suggest that the chromosome orders within the two haploid sets are antiparallel. This chromosome arrangement in human cells appears to be both independent of cell type- and species-specific and may influence chromosome topology throughout the cell cycle.