Phosphoryl transfer step in the C-terminal Src kinase controls Src recognition

All members of the Src family of nonreceptor protein tyrosine kinases are phosphorylated and subsequently down-regulated by the C-terminal Src kinase, Csk. Although the recognition of Src protein substrates is essential for a diverse set of signaling events linked to cellular growth and differentiation, the factors controlling this critical protein-protein interaction are not well known. To understand how Csk recognizes Src, the chemical/physical events that modulate apparent substrate affinity and turnover were investigated. Src is phosphorylated in a biphasic manner in rapid quench flow experiments, suggesting that the phosphoryl transfer step is fast and highly favorable and does not limit overall turnover. As opposed to other kinase-substrate pairs, turnover is not limited by the physical release of ADP based on stopped-flow fluorescence and catalytic trapping experiments, suggesting that other steps control net phosphorylation. The K(d) for Src is considerably larger than the K(m) based on single turnover kinetic and equilibrium sedimentation experiments. Taken together, the data are consistent with a mechanism whereby Csk achieves a low K(m) for the substrate Src, not by stabilizing protein-protein interactions but rather by facilitating a fast phosphoryl transfer step. In this manner, the phosphoryl transfer step functions as a chemical clamp facilitating substrate recognition.     

SRC tail phosphorylation is limited by structural changes in the regulatory tyrosine kinase Csk

Src family tyrosine kinases are down-regulated through phosphorylation of a single C-terminal tyrosine by the nonreceptor tyrosine kinase Csk. Despite the fundamental role of Csk in controlling cell growth and differentiation, it is unclear what limits this key signaling reaction and controls the production of catalytically repressed Src. To investigate this issue, stopped-flow fluorescence experiments were performed to determine which steps modulate catalysis. Both Src binding and phosphorylation can be monitored by changes in intrinsic tryptophan fluorescence. Association kinetics are biphasic with the initial phase corresponding to the bimolecular interaction of both proteins and the second phase representing a slow conformational change that coincides with the rate of maximum turnover. The kinetic transients for the phosphorylation reaction are also biphasic with the initial phase corresponding to the rapid phosphorylation and the release of phospho-Src. These data, along with equilibrium sedimentation and product inhibition experiments, suggest that steps involving Src association, phosphorylation, and product release are fast and that a structural change in Csk participates in limiting the catalytic cycle.     

Molecular determinants for Csk-catalyzed tyrosine phosphorylation of the Src tail

Phosphorylation of a critical tail tyrosine residue in Src modulates its three-dimensional structure and protein tyrosine kinase activity. The protein tyrosine kinase Csk is responsible for catalyzing the phosphorylation of this key Src tyrosine residue, but the detailed molecular basis for Src recognition and catalysis is poorly understood. In this study, we investigate this phosphorylation event using purified recombinant Csk and Src proteins and mutants. It was shown that the apparent k(cat) and K(m) values for Csk phosphorylation of catalytically impaired Src (dSrc) are similar to the parameters for Csk-catalyzed phosphorylation of the Src family member Lck. The SH3 (Src homology 3) and SH2 (Src homology 2) domains of dSrc were fully dispensable with respect to rapid phosphorylation, indicating that the catalytic domain and tail of dSrc are sufficient for the high efficiency of dSrc as a substrate. Of the eight Src tail residues examined, only the fully conserved Glu (Y-3 position) and Gln (Y-1 position) investigated by alanine scanning mutagenesis caused large reductions (10--40-fold) in dSrc substrate efficiency. The Y-3 Glu requirement was stringent as conservative replacements with Asp or Gln were no better than Ala whereas replacement of the Y-1 Gln with Ile was readily tolerated. Interestingly, en bloc replacement of the tail with a seven amino acid consensus sequence derived from a peptide library analysis was no better than the wild-type sequence. Surprisingly, the dSrc Y527F protein, although not a Csk substrate, enhanced Csk-catalyzed phosphorylation of dSrc. These results and other data suggest that Src dimerization (or higher order oligomerization) is important for high-efficiency Csk-catalyzed phosphorylation of the Src tail.     

Domain interactions in protein tyrosine kinase Csk.

Csk (C-terminal Src kinase) is a protein tyrosine kinase that phosphorylates Src family member C-terminal tails, resulting in downregulation of Src family members. It is composed of three principal domains: an SH3 (Src homology 3) domain, an SH2 (Src homology 2) domain, and a catalytic domain. The impact of the noncatalytic domains on kinase catalysis was investigated. The Csk catalytic domain was expressed in Escherichia coli as a recombinant glutathione S-transferase-fusion protein and demonstrated to have 100-fold reduced catalytic efficiency. Production of the catalytic domain by proteolysis of full-length Csk afforded a similar rate reduction. This suggested that the reduction in catalytic efficiency of the recombinant catalytic domain was intrinsic to the sequence and not an artifact related to faulty expression. This rate reduction was similar for peptide and protein substrates and was due almost entirely to a reduced k(cat) rather than to effects on substrate K(m)s. Viscosity experiments on the catalytic fragment kinase reaction demonstrated that the chemical (phosphoryl transfer) step had a reduced rate. While the Csk SH2 domain had no intermolecular effect on the kinase activity of the Csk catalytic domain, the SH3 domain and SH3-SH2 fragment led to a partial rescue (4-5-fold) of the lost kinase activity. This rescue was not achieved with two other SH3 domains (lymphoid cell kinase, Abelson kinase). The extrapolated K(d) of interaction for the Csk catalytic domain with the Csk SH3 domain was 2.2 microM and that of the Csk catalytic domain with the Csk SH3-SH2 fragment was 8.8 microM. Taken together, these findings suggest that there is likely an intramolecular interaction between the catalytic and SH3 domains in full-length Csk that is important for efficient catalysis. By employing a Csk SH3 specific type II polyproline helix peptide and carrying out site-directed mutagenesis, it was established that the SH3 surface that interacts with the catalytic domain was distinct from the surface that binds type II polyproline helix peptides. This finding suggests a novel mode of protein-protein interaction for an SH3 domain. The implications for Csk substrate selectivity, regulation, and function are discussed.     

Two rate-limiting steps in the kinetic mechanism of the serine/threonine specific protein kinase ERK2: a case of fast phosphorylation followed by fast product release

Extracellular regulated protein kinase 2 (ERK2) is a eukaryotic protein kinase whose activity is regulated by mitogenic stimuli. To gain insight into the catalytic properties of ERK2 and to complement structure-function studies, we undertook a pre-steady state kinetic analysis of the enzyme. To do this, ERK2 was quantitatively activated by MAPKK1 in vitro by monitoring the stoichiometry and site specificity of phosphorylation using a combination of protein mass spectrometry, tryptic peptide analysis, and (32)P radiolabeling. Using a quench-flow apparatus, MgATP(2-) was rapidly mixed (<1 ms) with both ERK2 and the protein substrate EtsDelta138 in the presence of a saturating total concentration (20 mM) of magnesium ion at 27 degrees C and pH 7.5. An exponential burst of product was observed over the first few milliseconds that followed mixing. This burst had an amplitude alpha of 0.44 and was followed by a slower linear phase. The pre-steady state burst is consistent with two partially rate-limiting enzymatic steps, which have the following rate constants: k(2) = 109 +/- 9 s(-1) and k(3) = 56 +/- 4 s(-1). These are attributed to rapid phosphorylation of EtsDelta138 and the process of product release, respectively. Single-turnover experiments provided an independent determination of k(2) (106 +/- 25 s(-1)). The observed catalytic constant (k(cat)(obs)) was found to be sensitive to the concentration of ERK2. The data fit a model in which ERK2 monomers form dimers and suggest that both the monomeric and dimeric forms of ERK2 are active with catalytic constants (k(cat)) of 25 and 37 s(-1), respectively. In addition, the model suggests that in the presence of saturating concentrations of both magnesium and substrates ERK2 subunits dissociate with a dissociation constant (K(d)) of 32 +/- 16 nM.     

Chemical rescue of a mutant protein-tyrosine kinase

Protein-tyrosine kinases contain a catalytic loop Arg residue located either two or four positions downstream of a highly conserved Asp residue. In this study, the role of this Arg (Arg-318) in the protein-tyrosine kinase C-terminal Src kinase (Csk) was investigated. The observed k(cat) for phosphorylation of the random copolymer poly(Glu,Tyr) substrate by Csk R318A is approximately 3000-fold smaller compared with that of wild type Csk, whereas the K(m) values for ATP and poly(Glu,Tyr) are only mildly affected. The k(cat) value for poly(Glu,Tyr) phosphorylation by the Csk double mutant A316R,R318A is 100-fold greater than the k(cat) value for the single R318A mutant, suggesting that an Arg positioned at the alternative location fulfills a similar function as in wild type. Csk R318A kinase activity can also be partially recovered by several exogenous small molecules including guanidinium and imidazole. These molecules contain key features whose roles in catalysis can be rationalized from a known x-ray structure of the insulin receptor tyrosine kinase. Imidazole is the best of these activators, enhancing phosphorylation rates by Csk R318A up to 100-fold for poly(Glu,Tyr) and significantly stimulating Csk R318A phosphorylation of the physiologic substrate Src. This chemical rescue of mutant protein kinase activity might find applications in cell signal transduction experiments.     

Subdomain switching reveals regions that harbor substrate specificity and regulatory properties of protein tyrosine kinases

Csk and Src are two protein tyrosine kinases that share a similar overall multidomain structural organization and a high degree of sequence homology but have different substrate specificities and regulatory properties. In this study, we generated chimeric kinases of Csk and Src by switching the C-terminal lobes of their catalytic domains, and we characterized their substrate specificity and regulatory properties. First, both Csk and Src phosphorylate Src as a common substrate, but on different Tyr residues. The C-terminal lobes of the kinase catalytic domain determined the site of phosphorylation on Src. Furthermore, toward several physiological substrates of Src, the substrate specificity was also determined by the C-terminal lobe of the catalytic domain regardless of the regulatory domains and the N-terminal lobe of the catalytic domain. Second, Csk and Src represent two general regulatory strategies for protein tyrosine kinases. Csk catalytic domain is inactive and is positively regulated by the regulatory domains, while Src catalytic domain is active and suppressed by its interactions with the regulatory domains. The regulatory properties of the chimeric kinases were more complicated. The regulatory domains and the N-lobe did not fully determine the response to a regulatory ligand, suggesting that the C-lobe also contributes to such responses. On the other hand, the intrinsic kinase activity of the catalytic domain correlates with the identity of the N-lobe. These results demonstrate that the chimeric strategy is useful for detailed dissection of the mechanistic basis of substrate specificity and regulation of protein tyrosine kinases.     

Slow dissociation of ATP from the calcium ATPase

The acyl-phosphate intermediate of the sarcoplasmic reticulum calcium ATPase reaction, formed in a brief incubation of vesicular enzyme with 5 microM [gamma-32P]ATP and calcium, reacts biphasically with added ADP (pH 7.0, 25 degrees C, 100 mM KCl, 5 mM MgSO4). Both the burst size and the rate constant for the slow phase increase with increasing ADP concentration in the way that is expected if the burst represents very rapid formation of an equilibrium amount of enzyme-bound ATP and the slow phase represents rate-limiting dissociation of ATP. Also consistent with this interpretation are the slow labeling of phosphoenzyme under conditions in which unlabeled ATP must dissociate first and the observation of a burst of ATP formation on ADP addition to phosphoenzyme. Values of the equilibrium constants for ADP dissociation from phosphoenzyme (0.75 mM), for ATP formation on the enzyme (2.3), and for the ATP dissociation rate constant (37 s-1) were obtained from a quantitative analysis of the data.     

Probing the communication between the regulatory and catalytic domains of a protein tyrosine kinase, Csk

Protein tyrosine kinases (PTKs) are important regulators of mammalian cell function and their own activities are tightly regulated. Underlying their tight regulation, all PTKs contain multiple regulatory domains in addition to a catalytic domain. C-terminal Src kinase (Csk) contains a catalytic domain and a regulatory region, consisting of an SH3 and an SH2 domain. In this study, we probed the communication between the regulatory and catalytic domains of Csk. First, kinetic characterization of SH3 and SH2 domain deletion mutants demonstrated that the SH3 and SH2 domains were crucial in maintaining the full activity of Csk, but were not directly involved in Csk recognition of its physiological substrate, Src. Second, highly conserved Trp188, corresponding to a key residue in domain-domain communication in other PTKs, was found to be important for maintaining the active structure of Csk by the presence of the regulatory region, but not required for Csk activation triggered by a phosphopeptide binding to the SH2 domain. Third, structural alignment indicated that the presence of the regulatory domains modulated the conformation of multiple substructures in the catalytic domain, some directly and others remotely. Mutagenic and kinetic studies supported this assignment. This report extended previous studies of Csk domain-domain communication, and provided a foundation for further detailed investigation of this communication.     

Detection of conformational changes along the kinetic pathway of protein kinase A using a catalytic trapping technique.

The dissociation rate constants for the two products of the reaction catalyzed by protein kinase A, ADP and phosphopeptide, were measured using a catalytic trapping technique to determine the role of product release in enzyme turnover. The enzyme was preequilibrated with ADP, and the reaction was initiated with a peptide substrate, LRRASLG, and ATP in a rapid quench flow instrument. At high, free magnesium concentrations (>2 mM), the large 'burst' in phosphopeptide production disappears, and, at low concentrations of free magnesium (0.5-1 mM), the kinetic transients become sigmoidal prior to the linear turnover phase. Increasing the concentrations of ATP or ADP did not influence the shape of the kinetic transients in the first 20 ms. ADP preequilibration protects the enzyme from inhibition by the covalent inactivator p-fluorosulfonylbenzoyl 5'-adenosine at 0.5 mM free magnesium, indicating that a competent E. ADP complex forms at low metal concentrations and the sigmoidal behavior in the catalytic trapping experiment is not due to free enzyme at high ATP concentrations. Simulations of the data indicate that ADP release is rate-limiting for turnover at high magnesium concentrations, but, at lower physiological levels of 0.5 and 1 mM, the off rate of ADP is 3- and 2-fold higher than k(cat), respectively. In contrast, the initial portions of the kinetic transients at 0.5 mM free magnesium were unaffected by phosphopeptide preequilibration, indicating that the release rate of this product is significantly larger than turnover. The transient kinetic data, coupled with a previous report [Shaffer and Adams (1999) Biochemistry 38, 5572-5581], support a phosphorylation mechanism under physiological magnesium concentrations that incorporates two partially rate-determining conformational changes, one prior to and one after the phosphoryl transfer step. We propose that the initial step activates the enzyme through key positioning of one or more active-site residues and the second step relaxes this conformation, a prerequisite for a subsequent catalytic cycle.     

Structural basis for the recognition of c-Src by its inactivator Csk

The catalytic activity of the Src family of tyrosine kinases is suppressed by phosphorylation on a tyrosine residue located near the C terminus (Tyr 527 in c-Src), which is catalyzed by C-terminal Src Kinase (Csk). Given the promiscuity of most tyrosine kinases, it is remarkable that the C-terminal tails of the Src family kinases are the only known targets of Csk. We have determined the crystal structure of a complex between the kinase domains of Csk and c-Src at 2.9 A resolution, revealing that interactions between these kinases position the C-terminal tail of c-Src at the edge of the active site of Csk. Csk cannot phosphorylate substrates that lack this docking mechanism because the conventional substrate binding site used by most tyrosine kinases to recognize substrates is destabilized in Csk by a deletion in the activation loop.     

A phosphotyrosine-dependent protein interaction screen reveals a role for phosphorylation of caveolin-1 on tyrosine 14: recruitment of C-terminal Src kinase.

Caveolin-1 is a substrate for nonreceptor tyrosine kinases including Src, Fyn, and Abl. To investigate the function of caveolin-1 phosphorylation, we modified the Gal4-based yeast two-hybrid system to screen for phosphorylation-dependent protein interactions. A cDNA library was screened using the N terminus of caveolin-1 as bait in a yeast strain expressing the catalytic domain of Abl. We identified two proteins in this screen that interact with caveolin-1 in a phosphorylation-dependent manner: tumor necrosis factor-alpha receptor-associated factor 2 (TRAF2) and C-terminal Src kinase (Csk). TRAF2 bound to nonphosphorylated caveolin-1, but this association was increased 3-fold by phosphorylation. In contrast, association of Csk with caveolin-1 was completely dependent on phosphorylation of caveolin-1, both for fusion proteins in yeast (>35-fold difference in affinity) and for endogenous proteins in tissue culture cells. Our data suggest that phosphorylation of caveolin-1 leads to Csk translocation into caveolae. This may induce a feedback loop that leads to inactivation of the Src family kinases that are highly enriched in caveolae.     

An ATP-linked structural change in protein kinase A precedes phosphoryl transfer under physiological magnesium concentrations

The kinetic mechanism for the catalytic subunit of protein kinase A was evaluated using physiological concentrations of free magnesium (0.5 mM) and a rapid quench flow technique. When the enzyme is pre-equilibrated with ATP, the peptide substrate, LRRASLG (Kemptide), is phosphorylated in a biphasic manner with a rapid, exponential "burst" phase (kb) followed by a slower, linear phase (kL) that corresponds to the steady-state kinetic rate. Both the amplitude and the substrate-rate dependence of the initial, burst phase indicate that the rate of phosphoryl transfer is fast (approximately 500 s-1) and does not limit turnover (45 s-1). Viscosity studies indicate that, while Kemptide is in rapid equilibrium, ATP does not exchange rapidly with the active site and kcat/KATP is limited by the rate constant for nucleotide encounter. When the pre-steady-state kinetic experiments are initiated with ATP, a lag phase is observed at low ATP concentrations consistent with rate-limiting association. At high ATP concentrations (>1 mM), a burst phase is observed but the rate and amplitude are low on the basis of the bimolecular rate constant for ATP association and the rate constant for phosphoryl transfer. The kinetic data indicate that the phosphoryl transfer step is fast at physiological magnesium concentrations, but an ATP-linked conformational change precedes this step, limiting the burst phase rate constant. Simulations of the pre-steady-state kinetic transients indicate that turnover (45 s-1) is limited both by net product release (70 s-1) and by this structural change (170 s-1). This structural change may also occur at high free magnesium concentrations, but it must be significantly faster than 170 s-1 and, consequently, not rate-limiting for turnover (kcat = 20 s-1 at 10 mM free Mg2+). We propose that this conformational event is an obligatory component of the kinetic pathway and includes a movement of the catalytic residues necessary for supporting phosphoryl group donation.     

Phosphorylation of the calcium-transporting adenosinetriphosphatase by lanthanum ATP: rapid phosphoryl transfer following a rate-limiting conformational change

The calcium-transport ATPase (CaATPase) of rabbit sarcoplasmic reticulum preincubated with 0.02 mM Ca2+ (cE.Ca2) is phosphorylated upon the addition of 0.25 mM LaCl3 and 0.3 mM [gamma-32P]ATP with an observed rate constant of 6.5 s-1 (40 mM MOPS, pH 7.0, 100 mM KCl, 25 degrees C). La.ATP binds to cE.Ca2 with a rate constant of 5 X 10(6) M-1 s-1, while ATP, Ca2+, and La3+ dissociate from cE.Ca2.La.ATP at less than or equal to 1 s-1. The reaction of ADP with phosphoenzyme (EP) formed from La.ATP is biphasic. An initial rapid loss of EP is followed by a slower first-order disappearance, which proceeds to an equilibrium mixture of EP.ADP and nonphosphorylated enzyme with bound ATP. The fraction of EP that reacts in the burst (alpha) and the first-order rate constant for the slow phase (kb) increase proportionally with increasing concentrations of ADP to give maximum values of 0.34 and 65 s-1, respectively, at saturating ADP (KADPS = 0.22 mM). The burst represents rapid phosphoryl transfer and demonstrates that ATP synthesis and hydrolysis on the enzyme are fast. The phosphorylation of cE.Ca2 by La.ATP at 6.5 s-1 and the kinetics for the reaction of EP with ADP are consistent with a rate-limiting conformational change in both directions. The conformational change converts cE.Ca2.La.ATP to the form of the enzyme that is activated for phosphoryl transfer, aE.Ca2.La.ATP, at 6.5 s-1; this is much slower than the analogous conformational change at 220 s-1 with Mg2+ as the catalytic ion [Petithory & Jencks (1986) Biochemistry 25, 4493]. The rate constant for the conversion of aE.Ca2.La.ATP to cE.Ca2.La.ATP is 170 s-1. ATP does not dissociate measurably from aE.Ca2.La.ATP. Labeled EP formed from cE.Ca2 and La.ATP with leaky vesicles undergoes hydrolysis at 0.06 s-1. It is concluded that the reaction mechanism of the CaATPase is remarkably similar with Mg.ATP and La.ATP; however, the strong binding of La.ATP slows both the conformational change that is rate limiting for EP formation and the dissociation of La.ATP. An interaction between La3+ at the catalytic site and the calcium transport sites decreases the rate of calcium dissociation by greater than 60-fold. When cE-Ca2 is mixed with 0.3 mM ATP and 1.0 mM Cacl2, the phosphoenzyme is formed with an observed rate constant of 3 s-1. The phosphoenzyme formed from Ca.ATP reacts with 2.0 mM ADP and labeled ATP with a rate constant of 30 s-1; there may be a small burst (alpha less than or equal to 0.05).     

Docking-based substrate recognition by the catalytic domain of a protein tyrosine kinase, C-terminal Src kinase (Csk)

Protein tyrosine kinases are key enzymes of mammalian signal transduction. Substrate specificity is a fundamental property that determines the specificity and fidelity of signaling by protein tyrosine kinases. However, how protein tyrosine kinases recognize the protein substrates is not well understood. C-terminal Src kinase (Csk) specifically phosphorylates Src family kinases on a C-terminal Tyr residue, which down-regulates their activities. We have previously determined that Csk recognizes Src using a substrate-docking site away from the active site. In the current study, we identified the docking determinants in Src recognized by the Csk substrate-docking site and demonstrated an interaction between the docking determinants of Src and the Csk substrate-docking site for this recognition. A similar mechanism was confirmed for Csk recognition of another Src family kinase, Yes. Although both Csk and MAP kinases used docking sites for substrate recognition, their docking sites consisted of different substructures in the catalytic domain. These results helped establish a docking-based substrate recognition mechanism for Csk. This model may provide a framework for understanding substrate recognition and specificity of other protein tyrosine kinases.     

Interactions between the Fyn SH3-domain and adaptor protein Cbp/PAG derived ligands, effects on kinase activity and affinity

Csk-binding protein/phosphoprotein associated with glycosphingolipid-enriched domains is a transmembrane adaptor protein primarily involved in negative regulation of T-cell activation by recruitment of C-terminal Src kinase (Csk), a protein tyrosine kinase which represses Src kinase activity through C-terminal phosphorylation. Recruitment of Csk occurs via SH2-domain binding to PAG pTyr317, thus, the interaction is highly dependent on phosphorylation performed by the Src family kinase Fyn, which docks onto PAG using a dual-domain binding mode involving both SH3- and SH2-domains of Fyn. In this study, we investigated Fyn SH3-domain binding to 14-mer peptide ligands derived from Cbp/PAG-enriched microdomains sequence using biochemical, biophysical and computational techniques. Interaction kinetics and dissociation constants for the various ligands were determined by SPR. The local structural impact of ligand association has been evaluated using CD, and molecular modelling has been employed to investigate details of the interactions. We show that data from these investigations correlate with functional effects of ligand binding, assessed experimentally by kinase assays using full-length PAG proteins as substrates. The presented data demonstrate a potential method for modulation of Src family kinase tyrosine phosphorylation through minor changes of the substrate SH3-interacting motif.     

The Tyrosine Kinase Csk Dimerizes through Its SH3 Domain

The Src family kinases possess two sites of tyrosine phosphorylation that are critical to the regulation of kinase activity. Autophosphorylation on an activation loop tyrosine residue (Tyr 416 in commonly used chicken c-Src numbering) increases catalytic activity, while phosphorylation of a C-terminal tyrosine (Tyr 527 in c-Src) inhibits activity. The latter modification is achieved by the tyrosine kinase Csk (C-terminal Src Kinase), but the complete inactivation of the Src family kinases also requires the dephosphorylation of the activation loop tyrosine. The SH3 domain of Csk recruits the tyrosine phosphatase PEP, allowing for the coordinated inhibition of Src family kinase activity. We have discovered that Csk forms homodimers through interactions mediated by the SH3 domain in a manner that buries the recognition surface for SH3 ligands. The formation of this dimer would therefore block the recruitment of tyrosine phosphatases and may have important implications for the regulation of Src kinase activity.     

Substrate conformational restriction and CD45-catalyzed dephosphorylation of tail tyrosine-phosphorylated Src protein

Hydrolysis of the tail phosphotyrosine in Src family members is catalyzed by the protein-tyrosine phosphatase CD45, activating Src family-related signaling pathways. Using purified recombinant phospho-Src (P-Src) (amino acid residues 83-533) and purified recombinant CD45 catalytic (cytoplasmic) domain (amino acid residues 565-1268), we have analyzed the kinetic behavior of dephosphorylation. A time course of phosphatase activity showed the presence of a burst phase. By varying the concentration of P-Src, it was shown that the amplitude of this burst phase increased linearly with respect to P-Src concentration. Approximately 2% of P-Src was shown to be rapidly dephosphorylated followed by a slower linear phase. A P-Src protein substrate containing a functional point mutation in the Src homology domain 2 (SH2) led to more rapid dephosphorylation catalyzed by CD45, and this reaction showed only a single linear kinetic phase. These results were interpreted in terms of a model in which P-Src exists in a relatively slow dynamic equilibrium between "closed" and "open" conformational forms. Combined mutations in the SH2 and SH3 domain or the addition of an SH3 domain ligand peptide enhanced the accessibility of P-Src to CD45 by biasing P-Src to a more open form. Consistent with this model, a phosphotyrosine peptide that behaved as an SH2 domain binding ligand showed approximately 100-fold greater affinity for unphosphorylated Src versus P-Src. Surprisingly, P-Src possessing combined SH3 and SH2 functional inactivating point mutations was dephosphorylated by CD45 more slowly compared with P-Src completely lacking SH3 and SH2 domains. Additional data suggest that the SH3 and SH2 domains can inhibit accessibility of the P-Src tail to CD45 by interactions other than direct phosphotyrosine binding by the SH2 domain. Taken together, these results suggest how activation of Src family member signaling pathways by CD45 may be influenced by the presence or absence of ligand interactions remote from the tail.     

Kinetic analyses of a truncated mammalian myosin I suggest a novel isomerization event preceding nucleotide binding

MI(1IQ) is a complex of calmodulin and an epitope-tagged 85-kDa fragment representing the amino-terminal catalytic motor domain and the first of 6 calmodulin-binding IQ domains of the mammalian myosin I gene, rat myr-1 (130-kDa myosin I or MI(130)). We have determined the transient kinetic parameters that dictate the ATP hydrolysis cycle of mammalian myosin I by examining the properties of MI(1IQ). Transient kinetics reveal that the affinity of MI(1IQ) for actin is 12 nm. The ATP-induced dissociation of actin-MI(1IQ) is biphasic. The fast phase is dependent upon [ATP], whereas the slow phase is not; both phases show a Ca(2+) sensitivity. The fast phase is eliminated by the addition of ADP, 10 micrometer being required for half-saturation of the effect in the presence of Ca(2+) and 3 micrometer ADP in the absence of Ca(2+). The slow phase shares the same rate constant as ADP release (8 and 3 s(-)(1) in the presence and absence of Ca(2+), respectively), but cannot be eliminated by decreasing [ADP]. We interpret these results to suggest that actin-myosin I exists in two forms in equilibrium, one of which is unable to bind nucleotide. These results also indicate that the absence of the COOH-terminal 5 calmodulin binding domains of myr-1 do not influence the kinetic properties of MI(130) and that the Ca(2+) sensitivity of the kinetics are in all likelihood due to Ca(2+) binding to the first IQ domain.     

C-terminal Src Kinase (Csk)-mediated Phosphorylation of Eukaryotic Elongation Factor 2 (eEF2) Promotes Proteolytic Cleavage and Nuclear Translocation of eEF2

Protein-tyrosine kinase C-terminal Src kinase (Csk) was originally purified as a kinase for phosphorylating Src and other Src family kinases. The phosphorylation of a C-terminal tyrosine residue of Src family kinases suppresses their kinase activity. Therefore, most physiological studies regarding Csk function have been focused on Csk as a negative regulator of Src family tyrosine kinases and as a potential tumor suppressor. Paradoxically, the protein levels of Csk were elevated in some human carcinomas. In this report, we show that eukaryotic elongation factor 2 (eEF2) is a new protein substrate of Csk and could locate in the nucleus. We demonstrate that Csk-mediated phosphorylation of eEF2 has no effect on its cytoplasmic function in regulating protein translation. However, phosphorylation of eEF2 enhances its proteolytic cleavage and the nuclear translocation of the cleaved eEF2 through a SUMOylation-regulated process. Furthermore, we show that cleaved fragments of eEF2 can induce nuclear morphological changes and aneuploidy similar to those in cancer cells, suggesting that there is an additional mechanism for Csk in tumorigenesis through regulation of eEF2 subcellular localization.