Reciprocal regulation of Hck activity by phosphorylation of Tyr(527) and Tyr(416). Effect of introducing a high affinity intramolecular SH2 ligand

The Src family tyrosine kinase Hck possesses two phosphorylation sites, Tyr(527) and Tyr(416), that affect the catalytic activity in opposite ways. When phosphorylated, Tyr(527) and residues C-terminal to it are involved in an inhibitory intramolecular interaction with the SH2 domain. However, this sequence does not conform to the sequence of the high affinity SH2 ligand, pYEEI. We mutated this sequence to YEEI and show that this mutant form of Hck cannot be activated by exogenous SH2 ligands. The SH3 domain of Hck is also involved in an inhibitory interaction with the catalytic domain. The SH3 ligand Nef binds to and activates YEEI-Hck mutant in a similar manner to wild-type Hck, indicating that disrupting the SH3 interaction overrides the strengthened SH2 interaction. The other phosphorylation site, Tyr(416), is the autophosphorylation site in the activation loop. Phosphorylation of Tyr(416) is required for Hck activation. We mutated this residue to alanine and characterized its catalytic activity. The Y416A mutant shows a higher K(m) value for peptide and a lower V(max) than autophosphorylated wild-type Hck. We also present evidence for cross-talk between the activation loop and the intramolecular binding of the SH2 and SH3 domains.     

c-Abl has high intrinsic tyrosine kinase activity that is stimulated by mutation of the Src homology 3 domain and by autophosphorylation at two distinct regulatory tyrosines

Using the specific Abl tyrosine kinase inhibitor STI 571, we purified unphosphorylated murine type IV c-Abl and measured the kinetic parameters of c-Abl tyrosine kinase activity in a solution with a peptide-based assay. Unphosphorylated c-Abl exhibited substantial peptide kinase activity with K(m) of 204 microm and V(max) of 33 pmol min(-1). Contrary to previous observations using immune complex kinase assays, we found that a transforming c-Abl mutant with a Src homology 3 domain point mutation (P131L) had significantly (about 6-fold) higher intrinsic kinase activity than wild-type c-Abl (K(m) = 91 microm, V(max) = 112 pmol min(-1)). Autophosphorylation stimulated the activity of wild-type c-Abl about 18-fold and c-Abl P131L about 3.6-fold, resulting in highly active kinases with similar catalytic rates. The autophosphorylation rate was dependent on Abl protein concentration consistent with an intermolecular reaction. A tyrosine to phenylalanine mutation (Y412F) at the c-Abl residue homologous to the c-Src catalytic domain autophosphorylation site impaired the activation of wild-type c-Abl by 90% but reduced activation of c-Abl P131L by only 45%. Mutation of a tyrosine (Tyr-245) in the linker region between the Src homology 2 and catalytic domains that is conserved among the Abl family inhibited the autophosphorylation-induced activation of wild-type c-Abl by 50%, whereas the c-Abl Y245F/Y412F double mutant was minimally activated by autophosphorylation. These results support a model where c-Abl is inhibited in part through an intramolecular Src homology 3-linker interaction and stimulated to full catalytic activity by sequential phosphorylation at Tyr-412 and Tyr-245.     

Src homology domains of v-Src stabilize an active conformation of the tyrosine kinase catalytic domain

To examine the interactions between Src homology,domains and the tyrosine kinase catalytic domain of v-Src, various combinations of domains have been expressed in bacteria as fusion proteins. Constructs containing the isolated catalytic domain, SH2 + catalytic domain, and SH3 + SH2 + catalytic domains were active in autophosphorylation assays. For the catalytic domain of v-Src, but not for v-Abl, addition of exogenous Src SH3-SH2 domains stimulated the autophosphorylation activity. In contrast to results for autophosphorylation, constructs containing Src homology domains were more active towards a synthetic peptide substrate than the isolated catalytic domain. The ability of the SH2 and SH3 domains of v-Src to stabilize an active enzyme conformation was also confirmed by refolding after denaturation in guanidinium hydrochloride. Collectively the data suggest that, in addition to their roles in intermolecular protein-protein interactions, the Src homology regions of v-Src exert a positive influence on tyrosine kinase function, potentially by maintaining an active conformation of the catalytic domain.     

PSM/SH2B1 splice variants: critical role in src catalytic activation and the resulting STAT3s-mediated mitogenic response

A role of PSM/SH2B1 had been shown in mitogenesis and extending to phenotypic cell transformation, however, the underlying molecular mechanism remained to be established. Here, four alternative PSM splice variants and individual functional protein domains were compared for their role in the regulation of Src activity. We found that elevated cellular levels of PSM variants resulted in phenotypic cell transformation and potentiated cell proliferation and survival in response to serum withdrawal. PSM variant activity presented a consistent signature pattern for any tested response of highest activity observed for gamma, followed by delta, alpha, and beta with decreasing activity. PSM-potentiated cell proliferation was sensitive to Src inhibitor herbimycin and PSM and Src were found in the same immune complex. PSM variants were substrates of the Src Tyr kinase and potentiated Src catalytic activity by increasing the V(max) and decreasing the K(m) for ATP with the signature pattern of variant activity. Dominant-negative PSM peptide mimetics including the SH2 or PH domains inhibited Src catalytic activity as well as Src-mediated phenotypic cell transformation. Activation of major Src substrate STAT3 was similarly potentiated by the PSM variants in a Src-dependent fashion or inhibited by PSM domain-specific peptide mimetics. Expression of a dominant-negative STAT3 mutant blocked PSM variant-mediated phenotypic cell transformation. Our results implicate an essential role of the PSM variants in the activation of the Src kinase and the resulting mitogenic response--extending to phenotypic cell transformation and involving the established Src substrate STAT3.     

Direct interaction of v-Src with the focal adhesion kinase mediated by the Src SH2 domain.

The recently described focal adhesion kinase (FAK) has been implicated in signal transduction pathways initiated by cell adhesion receptor integrins and by neuropeptide growth factors. To examine the mechanisms by which FAK relays signals from the membrane to the cell interior, we carried out a series of experiments to detect potential FAK interactions with proteins containing Src homology 2 (SH2) domains that are important intracellular signaling molecules. Using v-Src-transformed NIH3T3 cells, we showed that FAK was present in the immune-complex precipitated by anti-Src antibody, suggesting potential interaction of FAK with v-Src in vivo. We also showed potentially direct interaction of FAK with v-Src in vivo using the yeast two-hybrid system. Using recombinant FAK expressed in insect cells and bacterial fusion proteins containing Src SH2 domains, we showed direct binding of FAK to the Src SH2 domain but not to the SH3 domain in vitro. A kinase-defective mutant of FAK, which is not autophosphorylated, did not interact with the Src SH2 domain under the same conditions, suggesting the involvement of the FAK autophosphorylation sites. Treatment of FAK with a protein-tyrosine phosphatase decreased its binding to the Src SH2 domain, whereas autophosphorylation in vitro increased its binding. These results confirm the importance of FAK autophosphorylation sites in its interaction with SH2 domain-containing proteins. Taken together, these results suggest that FAK may mediate signal transduction events initiated on the cell surface by kinase activation and autophosphorylation that result in its binding to other key intracellular signaling molecules.     

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.     

An intramolecular interaction between SH2-kinase linker and kinase domain is essential for the catalytic activity of protein-tyrosine kinase-6

Protein-tyrosine kinase-6 (PTK6, also known as Brk) is a non-receptor tyrosine kinase that contains SH3, SH2, and catalytic (Kinase) domains. We have identified an intramolecular interaction between the linker (Linker) region connecting the SH2 and Kinase domains and the Kinase domain. Residue Trp-184 within the Linker region is essential for the Linker-Kinase interaction but not for the Linker-SH3 interaction. A recombinant PTK6 Kinase domain connected to the Linker region had catalytic activity in terms of autophosphorylation, phosphorylation of a PTK6 substrate, BKS, and phosphorylation of an oligopeptide substrate, whereas the Kinase domain itself, or one connected to a Linker region containing a W184A substitution, did not. The introduction of the W184A mutation into PTK6 also abrogated autophosphorylation and phosphorylation of another PTK6 substrate, Sam68, as well as phosphorylation of intracellular proteins. It also abolished the ability of PTK6 to promote proliferation and prevent apoptosis of HEK 293 cells, as well as to permit anchorage-independent colony formation. Therefore, unlike Src family members, in which the Linker-Kinase interaction inhibits catalytic activity, in PTK6 this interaction has an essential positive role.     

Biochemical properties of the Cdc42-associated tyrosine kinase ACK1. Substrate specificity, authphosphorylation, and interaction with Hck

ACK1 (activated Cdc42-associated kinase 1) is a nonreceptor tyrosine kinase and the only tyrosine kinase known to interact with Cdc42. To characterize the enzymatic properties of ACK, we have expressed and purified active ACK using the baculovirus/Sf9 cell system. This ACK1 construct contains (from N to C terminus) the kinase catalytic domain, SH3 domain, and Cdc42-binding Cdc42/Rac interactive binding (CRIB) domain. We characterized the substrate specificity of ACK1 using synthetic peptides, and we show that the specificity of the ACK1 catalytic domain most closely resembles that of Abl. Purified ACK1 undergoes autophosphorylation, and autophosphorylation enhances kinase activity. We identified Tyr284 in the activation loop of ACK1 as the primary autophosphorylation site using mass spectrometry. When expressed in COS-7 cells, the Y284F mutant ACK1 showed dramatically reduced levels of tyrosine phosphorylation. Although the SH3 and CRIB domains of purified ACK1 are able to bind ligands (a polyproline peptide and Cdc42, respectively), the addition of ligands did not stimulate tyrosine kinase activity. To characterize potential interacting partners for ACK1, we screened several SH2 and SH3 domains for their ability to bind to full-length ACK1 or to the catalytic-SH3-CRIB construct. ACK1 interacts most strongly with the SH3 domains of Src family kinases (Src or Hck) via its C-terminal proline-rich domain. Co-expression of Hck with kinase-inactive ACK1(K158R) in mammalian cells resulted in tyrosine phosphorylation of ACK1, suggesting that ACK1 is a substrate for Hck. Our data suggest that Hck is a novel binding partner for ACK1 that can regulate ACK1 activity by phosphorylation.     

Purification and kinetics of a raw starch-hydrolyzing,thermostable, and neutral glucoamylase of the thermophilic mold Thermomucor indicae-seudaticae

The purified glucoamylase of the thermophilic mold Thermomucor indicae-seudaticaehad a molecular mass of 42 kDa with a pI of 8.2. It is a glycoprotein with 9-10.5% carbohydrate content, which acted optimally at 60 degrees C and pH 7.0, with a t(1/2) of 12 h at 60 degrees C and 7 h at 80 degrees C. Its experimental activation energy was 43 KJ mol(-1) with temperature quotient (Q(10)) of 1.35, while the values predicted by response surface methodology (RSM) were 43 KJ mol(-1) and 1.28, respectively. The enzyme hydrolyzed soluble starch at 50 degrees C (K(m) 0.50 mg mL(-1) and V(max) 109 micromol mg(-1) protein min(-1)) and at 60 degrees C (K(m) 0.40 and V(max) 143 micromol mg(-1) protein min(-1)). The experimental K(m) and V(max) values are in agreement with the predicted values at 50 degrees C (K(m) 0.45 mg mL(-1) and V(max) 111.11 micromol mg(-1) protein min(-1)) and at 60 degrees C (K(m) 0.36 mg mL(-1)and V(max) 142.85 micromol mg(-1) protein min(-1)). An Arrhenius plot indicated thermal activation up to 60 degrees C, and thereafter, inactivation. The enzyme was strongly stimulated by Co(2+), Fe(2+), Ag(2+), and Ca(2+), slightly stimulated by Cu(2+) and Mg(2+), and inhibited by Hg(2+), Zn(2+), Ni(2+), and Mn(2+). Among additives, dextran and trehalose slightly enhanced the activity. Glucoamylase activity was inhibited by EDTA, beta-mercaptoethanol, dithiothreitol, and n-bromosuccinimide, and n-ethylmaleimide inhibited its activity completely. This suggested the involvement of tryptophan and cysteine in catalytic activity and the critical role of disulfide linkages in maintaining the conformation of the enzyme. The enzyme hydrolyzed around 82% of soluble starch and 65% of raw starch (K(m) 2.4 mg mL(-1), V(max) 50 micromol mg(-1) protein min(-1)), and it was remarkably insensitive to glucose, suggesting its applicability in starch saccharification.     

Src protein-tyrosine kinase structure and regulation

Src and Src-family protein kinases are proto-oncogenes that play key roles in cell morphology, motility, proliferation, and survival. v-Src (a viral protein) is encoded by the chicken oncogene of Rous sarcoma virus, and Src (the cellular homologue) is encoded by a physiological gene, the first of the proto-oncogenes. From the N- to C-terminus, Src contains an N-terminal 14-carbon myristoyl group, a unique segment, an SH3 domain, an SH2 domain, a protein-tyrosine kinase domain, and a C-terminal regulatory tail. The chief phosphorylation sites of Src include tyrosine 416 that results in activation from autophosphorylation and tyrosine 527 that results in inhibition from phosphorylation by C-terminal Src kinase. In the restrained state, the SH2 domain forms a salt bridge with phosphotyrosine 527, and the SH3 domain binds to the kinase domain via a polyproline type II left-handed helix. The SH2 and SH3 domains occur on the backside of the kinase domain away from the active site where they stabilize a dormant enzyme conformation. Protein-tyrosine phosphatases such as PTPalpha displace phosphotyrosine 527 from the Src SH2 domain and mediate its dephosphorylation leading to Src kinase activation. C-terminal Src kinase consists of an SH3, SH2, and kinase domain; it lacks an N-terminal myristoyl group and a C-terminal regulatory tail. Its X-ray structure has been determined, and the SH2 lobe occupies a position that is entirely different from that of Src. Unlike Src, the C-terminal Src kinase SH2 and SH3 domains stabilize an active enzyme conformation. Amino acid residues in the alphaD helix near the catalytic loop in the large lobe of C-terminal Src kinase serve as a docking site for the physiological substrate (Src) but not for an artificial substrate (polyGlu(4)Tyr).     

Mechanism and functional significance of Itk autophosphorylation

Tec family non-receptor tyrosine kinases (Itk, Btk, Tec, Rlk and Bmx) are characterized by the presence of an autophosphorylation site within the non-catalytic Src homology 3 (SH3) domain. The full-length Itk mutant containing phenylalanine in place of the autophosphorylated tyrosine has been studied in Itk-deficient primary T cells. These studies revealed that the non-phosphorylated enzyme restores Itk mediated signaling only partially. In spite of these insights, the precise role of the Tec kinase autophosphorylation site is unclear and the mechanism of the autophosphorylation reaction within the Tec kinases is not known. Here, we show both in vitro and in vivo that Itk autophosphorylation on Y180 within the SH3 domain occurs exclusively via an intramolecular, in cis mechanism. Using an in vitro kinase assay, we show that mutation of the Itk autophosphorylation site Y180 to Phe decreases kinase activity of the full-length enzyme by increasing Km for a peptide substrate. Moreover, mutation of Y180 to Glu, a residue chosen to mimic the phosphorylated tyrosine, alters the ligand-binding capability of the Itk SH3 domain in a ligand-dependent fashion. NMR chemical shift mapping gives residue-specific structural insight into the effect of the Y180E mutation on ligand binding. These data provide a molecular level context with which to interpret in vivo functional data and allow development of a structural model for Itk autophosphorylation.     

ATP-phosphopeptide conjugates as inhibitors of Src tyrosine kinases

A number of Src SH2 domain inhibitors enhance the kinase catalytic activity by switching the closed inactive to the open active conformation. ATP-phosphopeptide conjugates were designed and synthesized as Src tyrosine kinase inhibitors based on a tetrapeptide sequence pTyr-Glu-Glu-Ile (pYEEI) and ATP to block the SH2 domain signaling and substrate phosphorylation by ATP, respectively. In general, ATP-phosphopeptide conjugates with optimal linkers such as compounds 5 and 7 (K(i) = 1.7-2.6 microM) showed higher binding affinities to the ATP-binding site relative to the other ATP-phosphopeptide conjugates having short or long linkers, 1-4 and 6, (K(i) = 10.1-16.1 microM) and ATP (K(m) = 74 microM). These ATP-phosphopeptide conjugates may serve as novel templates for designing protein tyrosine kinase inhibitors to block SH2 mediated protein-protein interactions and to counter the activation of enzyme that resulted from the SH2 inhibition.     

The Cdc42-associated kinase ACK1 is not autoinhibited but requires Src for activation

The non-RTK (receptor tyrosine kinase) ACK1 [activated Cdc42 (cell division cycle 42)-associated kinase 1] binds a number of RTKs and is associated with their endocytosis and turnover. Its mode of activation is not well established, but models have suggested that this is an autoinhibited kinase. Point mutations in its SH3 (Src homology 3)- or EGF (epidermal growth factor)-binding domains have been reported to activate ACK1, but we find neither of the corresponding W424K or F820A mutations do so. Indeed, deletion of the various ACK1 domains C-terminal to the catalytic domain are not associated with increased activity. A previous report identified only one major tyrosine phosphorylated protein of 60 kDa co-purified with ACK1. In a screen for new SH3 partners for ACK1 we found multiple Src family kinases; of these c-Src itself binds best. The SH2 and SH3 domains of Src interact with ACK1 Tyr518 and residues 623-652 respectively. Src targets the ACK1 activation loop Tyr284, a poor autophosphorylation site. We propose that ACK1 fails to undergo significant autophosphorylation on Tyr284 in vivo because it is basophilic (whereas Src is acidophilic). Subsequent ACK1 activation downstream of receptors such as EGFR (EGF receptor) (and Src) promotes turnover of ACK1 in vivo, which is blocked by Src inhibitors, and is compromised in the Src-deficient SYF cell line. The results of the present study can explain why ACK1 is responsive to so many external stimuli including RTKs and integrin ligation, since Src kinases are commonly recruited by multiple receptor systems.     

Purification and characterization of a Ca(2+)-dependent protein kinase from sandalwood (Santalum album L.): evidence for Ca(2+)-induced conformational changes

An early development-specific soluble 55 kDa Ca(2+)-dependent protein kinase has been purified to homogeneity from sandalwood somatic embryos and biochemically characterized. The purified enzyme, swCDPK, resolved into a single band on 10% polyacrylamide gels, both under denaturing and non-denaturing conditions. swCDPK activity was strictly dependent on Ca(2+), K(0.5) (apparent binding constant) for Ca(2+)-activation of substrate phosphorylation activity being 0.7 microM and for autophosphorylation activity approximately 50 nM. Ca(2+)-dependence for activation, CaM-independence, inhibition by CaM-antagonist (IC(50) for W7=6 microM, for W5=46 microM) and cross-reaction with polyclonal antibodies directed against the CaM-like domain of soybean CDPK, confirmed the presence of an endogenous CaM-like domain in the purified enzyme. Kinetic studies revealed a K(m) value of 1.3 mg/ml for histone III-S and a V(max) value of 0.1 nmol min(-1) mg(-1). The enzyme exhibited high specificity for ATP with a K(m) value of 10 nM. Titration with calcium resulted in the enhancement of intrinsic emission fluorescence of swCDPK and a shift in the lambda(max) emission from tryptophan residues. A reduction in the efficiency of non-radiative energy transfer from tyrosine to tryptophan residues was also observed. These are taken as evidence for the occurrence of Ca(2+)-induced conformational change in swCDPK. The emission spectral properties of swCDPK in conjunction with Ca(2+) levels required for autophosphorylation and substrate phosphorylation help understand mode of Ca(2+) activation of this enzyme.     

Partial purification and characterization of almond seed lipase

A lipase was partially purified from the almond (Amygdalus communis L.) seed by ammonium sulfate fractionation and dialysis. Kinetics of the enzyme activity versus substrate concentration showed typical lipase behavior, with K(m) and V(max) values of 25 mM and 113.63 micromol min(-1) mg(-1) for tributyrin as substrate. All triglycerides were efficiently hydrolyzed by the enzyme. The partially purified almond seed lipase (ASL) was stable in the pH range of 6-9.5, with an optimum pH of 8.5. The enzyme was stable between 20 and 90 degrees C, beyond which it lost activity progressively, and exhibited an optimum temperature for the hydrolysis of soy bean oil at 65 degrees C. Based on the temperature activity data, the activation energy for the hydrolysis of soy bean oil was calculated as -5473.6 cal/mol. Soy bean oil served as good substrate for the enzyme and hydrolytic activity was enhanced by Ca(2+), Fe(2+), Mn(2+), Co(2+), and Ba(2+), but strongly inhibited by Mg(2+), Cu(2+), and Ni(2+). The detergents, sodiumdeoxicholate and Triton X-100 strongly stimulated enzyme activity while CTAB, DTAB, and SDS were inhibitors. Triton X-405 had no effect on lipase activity. The partially purified enzyme retained its activity for more than 6 months at -20 degrees C, beyond which it lost activity progressively.     

Ion selectivity and current saturation in inward-rectifier K+ channels

We investigated the features of the inward-rectifier K channel Kir1.1 (ROMK) that underlie the saturation of currents through these channels as a function of permeant ion concentration. We compared values of maximal currents and apparent K(m) for three permeant ions: K(+), Rb(+), and NH(4)(+). Compared with K(+) (i(max) = 4.6 pA and K(m) = 10 mM at -100 mV), Rb(+) had a lower permeability, a lower i(max) (1.8 pA), and a higher K(m) (26 mM). For NH(4)(+), the permeability was reduced more with smaller changes in i(max) (3.7 pA) and K(m) (16 mM). We assessed the role of a site near the outer mouth of channel in the saturation process. This site could be occupied by either permeant ions or low-affinity blocking ions such as Na(+), Li(+), Mg(2+), and Ca(2+) with similar voltage dependence (apparent valence, 0.15-0.20). It prefers Mg(2+) over Ca(2+) and has a monovalent cation selectivity, based on the ability to displace Mg(2+), of K(+) > Li(+) ～ Na(+) > Rb(+) ～ NH(4)(+). Conversely, in the presence of Mg(2+), the K(m) for K(+) conductance was substantially increased. The ability of Mg(2+) to block the channels was reduced when four negatively charged amino acids in the extracellular domain of the channel were mutated to neutral residues. The apparent K(m) for K(+) conduction was unchanged by these mutations under control conditions but became sensitive to the presence of external negative charges when residual divalent cations were chelated with EDTA. The results suggest that a binding site in the outer mouth of the pore controls current saturation. Permeability is more affected by interactions with other sites within the selectivity filter. Most features of permeation (and block) could be simulated by a five-state kinetic model of ion movement through the channel.     

SH2 domains of the p85 alpha subunit of phosphatidylinositol 3-kinase regulate binding to growth factor receptors.

The binding of cytoplasmic signaling proteins such as phospholipase C-gamma 1 and Ras GTPase-activating protein to autophosphorylated growth factor receptors is directed by their noncatalytic Src homology region 2 (SH2) domains. The p85 alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase, which associates with several receptor protein-tyrosine kinases, also contains two SH2 domains. Both p85 alpha SH2 domains, when expressed individually as fusion proteins in bacteria, bound stably to the activated beta receptor for platelet-derived growth factor (PDGF). Complex formation required PDGF stimulation and was dependent on receptor tyrosine kinase activity. The bacterial p85 alpha SH2 domains recognized activated beta PDGF receptor which had been immobilized on a filter, indicating that SH2 domains contact autophosphorylated receptors directly. Several receptor tyrosine kinases within the PDGF receptor subfamily, including the colony-stimulating factor 1 receptor and the Steel factor receptor (Kit), also associate with PI 3-kinase in vivo. Bacterially expressed SH2 domains derived from the p85 alpha subunit of PI 3-kinase bound in vitro to the activated colony-stimulating factor 1 receptor and to Kit. We infer that the SH2 domains of p85 alpha bind to high-affinity sites on these receptors, whose creation is dependent on receptor autophosphorylation. The SH2 domains of p85 are therefore primarily responsible for the binding of PI 3-kinase to activated growth factor receptors.     

SH2-Dependent Autophosphorylation within the Tec Family Kinase Itk

The Tec family kinase, Itk (interleukin-2 tyrosine kinase), undergoes an in cis autophosphorylation on Y180 within its Src homology 3 (SH3) domain. Autophosphorylation of the Itk SH3 domain by the Itk kinase domain is strictly dependent on the presence of the intervening Src homology 2 (SH2) domain. A direct docking interaction between the Itk kinase and SH2 domains brings the Itk SH3 domain into the active site where Y180 is then phosphorylated. We now identify the residues on the surface of the Itk SH2 domain responsible for substrate docking and show that this SH2 surface mediates autophosphorylation in the full-length Itk molecule. The canonical phospholigand binding site on the SH2 domain is not involved in substrate docking, instead the docking site consists of side chains from three loop regions (AB, EF and BG) and part of the βD strand. These results are extended into Btk (Bruton's tyrosine kinase), a Tec family kinase linked to the B-cell deficiency X-linked agammaglobulinemia (XLA). Our results suggest that some XLA-causing mutations might impair Btk phosphorylation.     

Src family protein tyrosine kinases induce autoactivation of Bruton's tyrosine kinase.

Bruton's tyrosine kinase (Btk) is tyrosine phosphorylated and enzymatically activated following ligation of the B-cell antigen receptor. These events are temporally regulated, and Btk activation follows that of various members of the Src family of protein tyrosine kinases, thus raising the possibility that Src kinases participate in the Btk activation process. We have evaluated the mechanism underlying Btk enzyme activation and have explored the potential regulatory relationship between Btk and Src protein kinases. We demonstrate in COS transient-expression assays that Btk can be activated through intramolecular autophosphorylation at tyrosine 551 and that Btk autophosphorylation is required for Btk catalytic functions. Coexpression of Btk with members of the Src family of protein tyrosine kinases, but not Syk, led to Btk tyrosine phosphorylation and activation. Using a series of point mutations in Blk (a representative Src protein kinase) and Btk, we show that Src kinases activate Btk through an indirect mechanism that requires membrane association of the Src enzymes as well as functional Btk SH3 and SH2 domains. Our results are compatible with the idea that Src protein tyrosine kinases contribute to Btk activation by indirectly stimulating Btk intramolecular autophosphorylation.     

A bioprocess for the production of phytase from Schizophyllum commune: studies of its optimization, profile of fermentation parameters, characterization and stability

Schizophyllum commune produces phytase through solid-state fermentation using different agroindustrial residues. After optimization of phytase production, a maximal level of phytase (113.7 Units/gram of dry substrate) was obtained in wheat bran based medium containing 5% sucrose, 50% humidity, 7.5% of biomass at 33 °C pH 7.0 during 72 h and a 285% improvement in enzyme titre was achieved. Analysis of fermentation parameters profile for phytase production showed the highest productivity (1.466 Units/gram of dry substrate/hour) in 66 h of fermentation. Phytase has an optimal pH of 5.0, an optimal temperature of 50 °C and K (m) and V (max) values of 0.16 mM and 1.85 μmol mL(-1) min(-1), respectively. Phytase activity was stimulated essentially in the presence of K(+), Ca(2+), Mg(2+), Mn(2+), Zn(2+), Cu(2+), Fe(2+), Fe(3+), Co(2+), Ni(2+), acetate and citrate at concentrations of 1 mM. Phytase had the best shelf life when stored at a cooling temperature, maintaining 38% of its initial activity after 112 days of storage, and still presenting enzymatic activity after 125 days of storage. Stability studies of phytase performed in aqueous enzyme extracts showed satisfactory results using polyethyleneglycol 3350, carboxymethylcellulose, methylparaben, mannitol and benzoic acid in concentrations of 0.25, 0.025, 0.025, 0.25, and 0.0025%, respectively. PEG 3350 was shown to be the best stabilizing agent, resulting in 109% of phytase activity from the initial crude extract remaining activity in after 90 days.