We have developed a method to study the primary sequence specificities of protein kinases by using an oriented degenerate peptide library. We report here the substrate specificities of eight protein Ser/Thr kinases. All of the kinases studied selected distinct optimal substrates. The identified substrate specificities of these kinases, together with known crystal structures of protein kinase A, CDK2, Erk2, twitchin, and casein kinase I, provide a structural basis for the substrate recognition of protein Ser/Thr kinases. In particular, the specific selection of amino acids at the ؉1 and ؊3 positions to the substrate serine/threonine can be rationalized on the basis of sequences of protein kinases. The identification of optimal peptide substrates of CDK5, casein kinases I and II, NIMA, calmodulin-dependent kinases, Erk1, and phosphorylase kinase makes it possible to predict the potential in vivo targets of these kinases.The essential role of protein kinases in regulating signal transduction was established with the discovery of cyclic AMPdependent protein kinase (PKA) (12). To respond to different extracellular stimuli, distinct groups of protein kinases have evolved. Each protein kinase is thought to phosphorylate a unique set of targets in the cell. The substrate specificities of protein kinases are therefore crucial for the fidelity of signaling events.The classical approach for studying the specificity of a protein kinase is to compare the phosphorylation kinetics of synthetic peptides on the basis of known sequences phosphorylated by the kinase. This procedure is helpful in identifying the amino acids critical for efficient phosphorylation. However, it is not practical to synthesize and study each of the billions of possible variations of sequences that must be considered. Moreover, it is extremely difficult to apply this approach to study the specificity of a protein kinase with no known substrates. To overcome these problems, we developed a method for determining the primary sequence specificities of protein kinases by using an oriented degenerate peptide library (21). Optimal peptide substrates of a given protein kinase are identified by phosphorylation of a pool of degenerate peptides containing billions of different species. The specificities determined for PKA, CDC2, and CDK2 by using this technique were consistent with known substrates of these kinases. The results also allowed the prediction of in vivo kinase substrates. Synthetic peptides based on predicted optimal motifs were shown to act as low-K m substrates for the kinases studied. Therefore, this method is a useful tool for studying substrate specificities of protein kinases.We present here the specificities of eight additional protein Ser/Thr kinases: CDK5, casein kinase I (CKI) ␦ and ␥, casein kinase II (CKII), NIMA, calmodulin-dependent (Cam) kinase II, Erk1, and phosphorylase kinase. Our findings demonstrate that each of these protein kinases has a distinct optimal peptide substrate. Critical determinants for recognition by the protein kinas...

The organization of cells into epithelium depends on cell interaction with both the extracellular matrix (ECM) and adjacent cells. The role of cell-cell adhesion in the regulation of epithelial topology is well-described. ECM is better known to promote cell migration and provide a structural scaffold for cell anchoring, but its contribution to multicellular morphogenesis is less well-understood. We developed a minimal model system to investigate how ECM affects the spatial organization of intercellular junctions. Fibronectin micropatterns were used to constrain the location of cell-ECM adhesion. We found that ECM affects the degree of stability of intercellular junction positioning and the magnitude of intra-and intercellular forces. Intercellular junctions were permanently displaced, and experienced large perpendicular tensional forces as long as they were positioned close to ECM. They remained stable solely in regions deprived of ECM, where they were submitted to lower tensional forces. The heterogeneity of the spatial organization of ECM induced anisotropic distribution of mechanical constraints in cells, which seemed to adapt their position to minimize both intra-and intercellular forces. These results uncover a morphogenetic role for ECM in the mechanical regulation of cells and intercellular junction positioning.pithelial sheets lie on a layer of extracellular matrix (ECM), the so-called basement membrane. In such epithelia, cells establish integrin-based adhesions on the basal part of the cell in contact with ECM, and cadherin-based intercellular adhesions on the apical part of contacting lateral domains, away from contact with ECM. The two adhesion systems display nonoverlapping spatial distributions. Both cell-cell and cell-ECM adhesions are required to establish proper epithelium morphology (1). They both participate in mechano-transduction of external physical cues into intracellular signaling (2). The biochemical nature of adhesion molecules engaged in intercellular adhesion, the energy of the interaction, as well as the mechanical tension developed along intercellular junctions have been shown to govern epithelial cell shape and orient intercellular junctions in various systems (3-6). However, whereas the contribution of cell-cell adhesion to epithelial topology has been the focus of many studies, much less attention has been paid to the role of ECM. ECM is a dynamic scaffold that is actively remodeled during morphogenesis, where it plays major roles in stimulating and guiding cell migration as well as orienting stem cell fate (7,8). ECM is also known to impart morphoregulatory signals to epithelia, and thereby regulates tissue morphogenesis (8, 9). However, the mechanism by which ECM guides cell positioning at the single-cell scale is still not known. ECM geometry has been shown to regulate intracellular architecture (10) and provide spatial information for cell polarization (1,11,12), but how it regulates cell positioning and thereby spatially organizes multicellular architectures remained to be i...

SummaryDopamine orchestrates motor behavior and reward-driven learning. Perturbations of dopamine signaling have been implicated in several neurological and psychiatric disorders, and in drug addiction. The actions of dopamine are mediated in part by the regulation of gene expression in the striatum, through mechanisms that are not fully understood. Here, we show that drugs of abuse, as well as natural reinforcement learning, promote the nuclear accumulation of dopamine-and cAMPregulated phosphoprotein Mr=32,000 . This accumulation is mediated through a signaling cascade involving dopamine D1 receptors, cAMP-dependent activation of protein phosphatase-2A, dephosphorylation of DARPP-32 at Ser-97 and inhibition of its nuclear export. The nuclear accumulation of DARPP-32, a potent inhibitor of protein phosphatase-1, increases phosphorylation of histone H3, an important component of nucleosomal response. Mutation of Ser-97 profoundly alters behavioral effects of drugs of abuse, and decreases motivation for food, underlining the functional importance of this signaling cascade.Midbrain dopamine (DA) neurons, activated following unexpected rewarding stimuli, are essential in reinforcement learning 1 . Drugs of abuse mimic the physiological action of DA neurons by increasing their firing rate or preventing DA uptake. Thus, they enhance extracellular DA levels in the forebrain, especially in the nucleus accumbens (NAc), a key structure required for the reinforcing effects of addictive drugs [2][3][4] . To understand how DA mediates reward-controlled learning, it is necessary to identify the intracellular events that trigger gene transcription alterations supporting long-lasting synaptic changes [5][6][7] . DARPP-32 (dopamine-and cAMP-regulated phosphoprotein, Mr=32,000) 8 is a prominent mediator of DA NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript signaling in the striatum 9 . DARPP-32 is highly enriched in striatal GABAergic mediumsize spiny neurons (MSN) 10 . Following activation of DA D1 receptors (D1R), DARPP-32 is phosphorylated by cAMP-dependent protein kinase (PKA) at Thr-34 and converted into a potent inhibitor of the multifunctional serine/threonine protein phosphatase-1 (PP1) 11 . DARPP-32-mediated inhibition of PP1 increases the phosphorylation of neurotransmitter receptors and ion channels crucial for synaptic function and plasticity 9 . DARPP-32 also regulates nuclear events, as demonstrated by alterations of drug-induced gene expression in mice lacking DARPP-32 or bearing a point mutation of 13 . Part of the control exerted by DARPP-32 on transcription is mediated by activation of the ERK pathway, dependent on the concomitant stimulation of D1R and glutamate NMDA receptors 13,14 . However, the precise mechanisms of information transfer from the cytoplasm to the nucleus of striatal neurons are still poorly characterized. Drugs of abuse and reinforcement learning trigger nuclear accumulation of DARPP-32 in striatal neuronsDARPP-32 has been extensively characterized as a cytoplasmic...

In tissues, cell microenvironment geometry and mechanics strongly impact on cell physiology. Surface micropatterning allows the control of geometry while deformable substrates of tunable stiffness are well suited for the control of the mechanics. We developed a new method to micropattern extracellular matrix proteins on poly-acrylamide gels in order to simultaneously control cell geometry and mechanics. Microenvironment geometry and mechanics impinge on cell functions by regulating the development of intra-cellular forces. We measured these forces in micropatterned cells. Micropattern geometry was streamlined to orient forces and place cells in comparable conditions. Thereby force measurement method could be simplified and applied to large-scale experiment on chip. We applied this method to mammary epithelial cells with traction force measurements in various conditions to mimic tumoral transformation. We found that, contrary to the current view, all transformation phenotypes were not always associated to an increased level of cell contractility.

Protein kinase CK2 is a tetramer composed of two α catalytic subunits and two β regulatory subunits. The structure of a C-terminal truncated form of the human β subunit has been determined by X-ray crystallography to 1.7 Å resolution. One dimer is observed in the asymmetric unit of the crystal. The most striking feature of the structure is the presence of a zinc finger mediating the dimerization. The monomer structure consists of two domains, one entirely α-helical and one including the zinc finger. The dimer has a crescent shape holding a highly acidic region at both ends. We propose that this acidic region is involved in the interactions with the polyamines and/or catalytic subunits. Interestingly, conserved amino acid residues among β subunit sequences are clustered along one linear ridge that wraps around the entire dimer. This feature suggests that protein partners may interact with the dimer through a stretch of residues in an extended conformation. Keywords: MAD method/protein kinase CK2/regulatory subunit/X-ray structure/zinc finger

X-ray crystallography studies, as well as live-cell fluorescent imaging, have recently challenged the traditional view of protein kinase CK2. Unbalanced expression of catalytic and regulatory CK2 subunits has been observed in a variety of tissues and tumours. Thus the potential intersubunit flexibility suggested by these studies raises the likely prospect that the CK2 holoenzyme complex is subject to disassembly and reassembly. In the present paper, we show evidence for the reversible multimeric organization of the CK2 holoenzyme complex in vitro. We used a combination of site-directed mutagenesis, binding experiments and functional assays to show that, both in vitro and in vivo, only a small set of primary hydrophobic residues of CK2beta which contacts at the centre of the CK2alpha/CK2beta interface dominates affinity. The results indicate that a double mutation in CK2beta of amino acids Tyr188 and Phe190, which are complementary and fill up a hydrophobic pocket of CK2alpha, is the most disruptive to CK2alpha binding both in vitro and in living cells. Further characterization of hotspots in a cluster of hydrophobic amino acids centred around Tyr188-Phe190 led us to the structure-based design of small-peptide inhibitors. One conformationally constrained 11-mer peptide (Pc) represents a unique CK2beta-based small molecule that was particularly efficient (i) to antagonize the interaction between the CK2 subunits, (ii) to inhibit the assembly of the CK2 holoenzyme complex, and (iii) to strongly affect its substrate preference.

The presence of fibroblast growth factor-2 (FGF-2) in the nucleus has now been reported both in vitro and in vivo, but its nuclear functions are unknown. Here, we show that FGF-2 added to nuclear extract binds to protein kinase CK2 and nucleolin, a CK2 natural substrate. Added to baculovirus-infected cell extracts overexpressing CK2 or its isolated subunits, FGF-2 binds to the enzyme through its regulatory ␤ subunit. Using purified proteins, FGF-2 is shown to directly interact with CK2 and to stimulate CK2 activity toward nucleolin. Furthermore, a mitogenic-deficient FGF-2 mutant protein has an impaired ability to interact with CK2 and to stimulate CK2 activity using nucleolin as substrate. We propose that in growing cells, one function of nuclear FGF-2 is to modulate CK2 activity through binding to its regulatory ␤ subunit.The fibroblast growth factors (FGFs) 1 family includes nine polypeptides of which FGF-1 and FGF-2 (acidic and basic FGF) are prototype members (reviewed in Refs. 1 and 2). FGF-2 exerts its pleiotropic effects in cell growth and differentiation through a dual receptor system consisting of four different high-affinity transmembrane receptor tyrosine kinases and low-affinity binding sites corresponding to heparan sulfate proteoglycans (reviewed in Refs. 2 and 3). In addition, FGF-1 and FGF-2 are translocated from outside the cell to the nucleus (reviewed in Ref.2). Many other growth factors have been detected in the cell nucleus (reviewed in Refs. 4 and 5). In the case of Schwannomaderived growth factor and FGF-1, nuclear localization is necessary for mitogenic activity (6 -8). For FGF-2, nuclear translocation is correlated to cell proliferation, since it is no longer recovered in the nucleus of confluent cells (9, 10). Therefore, these data strongly suggest that FGF-2, as well as other growth factors, plays specific, but still unknown, nuclear functions in addition to classical signaling through cell surface receptors.We reported previously that in synchronized ABAE growing cells, a large increase of ribosomal genes transcription is tightly correlated to the nuclear translocation of FGF-2 and especially to an accumulation of the growth factor in the nucleolus. In contrast, in quiescent cells ribosomal genes transcription is 20-fold lower, and FGF-2 is exclusively found in the cytoplasm (9, 10). Upon addition of FGF-2 to nuclei of serum-starved cells, a 6-fold increase of ribosomal genes transcription is observed together with an increase in phosphorylation of nuclear proteins, essentially of nucleolin (9, 11); These data suggest that a nuclear function of FGF-2 could be to regulate ribosomal genes transcription by modulating the phosphorylation level of nuclear and nucleolar proteins as nucleolin. Nucleolin that is the major component of nucleolus is also a major substrate for protein kinase CK2 in rapidly proliferating tissues, and its phosphorylation level is correlated to the rate of ribosome biogenesis (reviewed in Ref. 12). CK2 is a serine/threonine protein kinase present in both the cyt...

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