MBNL142 and MBNL143 gene isoforms, overexpressed in DM1-patient muscle, encode for nuclear proteins interacting with Src family kinases.

Myotonic dystrophy type-1 (DM1) is the most prevalent form of muscular dystrophy in adults. This disorder is an RNA-dominant disease, caused by expansion of a CTG repeat in the DMPK gene that leads to a misregulation in the alternative splicing of pre-mRNAs. The longer muscleblind-like-1 (MBNL1) transcripts containing exon 5 and the respective protein isoforms (MBNL142-43) were found to be overexpressed in DM1 muscle and localized exclusively in the nuclei. In vitro assays showed that MBNL142-43 bind the Src-homology 3 domain of Src family kinases (SFKs) via their proline-rich motifs, enhancing the SFK activity. Notably, this association was also confirmed in DM1 muscle and myotubes. The recovery, mediated by an siRNA target to Ex5-MBNL142-43, succeeded in reducing the nuclear localization of both Lyn and MBNL142-43 proteins and in decreasing the level of tyrosine phosphorylated proteins. Our results suggest an additional molecular mechanism in the DM1 pathogenesis, based on an altered phosphotyrosine signalling pathway.

Lyn-mediated SHP-1 recruitment to CD5 contributes to resistance to apoptosis of B-cell chronic lymphocytic leukemia cells.

In B-cell chronic lymphocytic leukemia (B-CLL) cells, Lyn, a tyrosine kinase belonging to the Src family, is overexpressed and atypically localized in an aberrant cytosolic complex in an active conformation, contributing to the unbalance between cell survival and pro-apoptotic signals. In this study, we demonstrate that Lyn constitutively phosphorylates the immunoreceptor tyrosine inhibitory motifs of the inhibitory cell surface co-receptor CD5, a marker of B-CLL. As a result, CD5 provides an anchoring site to Src homology 2 domain-containing phosphatase 1 (SHP-1), a known negative regulator of hematopoietic cell function, thereby triggering the negative B-cell receptor (BCR) signaling. The subsequent segregation of SHP-1 into two pools, one bound to the inhibitory co-receptor CD5 in an active form, the other in the cytosol in an inhibited conformation, proves crucial for withstanding apoptosis, as shown by the use of phosphotyrosine phosphatase-I-I, a direct inhibitor of SHP-1, or SHP-1 knockdown. These results confirm that Lyn exhibits the unique ability to negatively regulate BCR signaling, in addition to positively regulating effectors downstream of the BCR, and identify SHP-1 as a novel player in the deranged signaling network and as a potential attractive target for new therapeutic strategies in B-CLL.

Ca2+ -independent effects of spermine on pyruvate dehydrogenase complex activity in energized rat liver mitochondria incubated in the absence of exogenous Ca2+ and Mg2+.

In the absence of exogenous Ca(2+) and Mg(2+) and in the presence of EGTA, which favours the release of endogenous Ca(2+), the polyamine spermine is able to stimulate the activity of pyruvate dehydrogenase complex (PDC) of energized rat liver mitochondria (RLM). This stimulation exhibits a gradual concentration-dependent trend, which is maximum, about 140%, at 0.5 mM concentration, after 30 min of incubation. At concentrations higher than 0.5 mM, spermine still stimulates PDC, when compared with the control, but shows a slight dose-dependent decrease. Changes in PDC stimulation are very close to the phosphorylation level of the E(1alpha) subunit of PDC, which regulates the activity of the complex, but it is also the target of spermine. In other words, progressive dephosphorylation gradually enhances the stimulation of RLM and progressive phosphorylation slightly decreases it. These results provide the first evidence that, when transported in RLM, spermine can interact in various ways with PDC, showing dose-dependent behaviour. The interaction most probably takes place directly on a specific site for spermine on one of the regulatory enzymes of PDC, i.e. pyruvate dehydrogenase phosphatase (PDP). The interaction of spermine with PDC may also involve activation of another regulatory enzyme, pyruvate dehydrogenase kinase (PDK), resulting in an increase in E(1alpha) phosphorylation and consequently reduced stimulation of PDC at high polyamine concentrations. The different effects of spermine in RLM are discussed, considering the different activities of PDP and PDK isoenzymes. It is suggested that the polyamine at low concentrations stimulates the isoenzyme PDP(2) and at high concentrations it stimulates PDK(2).

Biological activity of antitumoural MGBG: the structural variable.

The present study aims at determining the structure-activity relationships (SAR's) ruling the biological function of MGBG (methylglyoxal bis(guanylhydrazone)), a competitive inhibitor of S-adenosyl-L-methionine decarboxylase displaying anticancer activity, involved in the biosynthesis of the naturally occurring polyamines spermidine and spermine. In order to properly understand its biochemical activity, MGBG's structural preferences at physiological conditions were ascertained, by quantum mechanical (DFT) calculations.

Analysis of a sub-proteome which co-purifies with and is phosphorylated by the Golgi casein kinase.

In an attempt to gain information about the identity of the Golgi apparatus casein kinase(s) (G-CK), responsible for the phosphorylation of caseins in lactating mammary gland, the proteins present in fractions enriched in G-CK activity eluted from DEAE-Sepharose and heparin-Sepharose columns were resolved by two-dimensional electrophoresis and analyzed by mass spectrometry. This led to the identification of 47 proteins altogether, none of which is a bona fide protein kinase. At least 9 of the identified proteins however, are readily phosphorylated by co-purifying G-CK activity, and 7 are physically associated with it to give supramolecular complex(es) of about 500 kDa as judged from Superdex S200 gel fitration and glycerol gradient ultracentrifugation experiments. In contrast, the apparent molecular weight of G-CK estimated from an in gel activity assay after SDSPAGE and renaturation is about 41 kDa. Many of the proteins phosphorylated by and/or associated with G-CK belong to the category of chaperonines, including HSP90, GRP-94 and -78, and various isoforms of protein disulfide isomerases, suggesting a global role of this kinase in the modulation of protein folding.

Tyrosine phosphatase activity in mitochondria: presence of Shp-2 phosphatase in mitochondria.

Tyrosine phosphorylation by unidentified enzymes has been observed in mitochondria, with recent evidence indicating that non-receptorial tyrosine kinases belonging to the Src family, which represent key players in several transduction pathways, are constitutively present in mitochondria. The extent of protein phosphorylation reflects a coordination balance between the activities of specific kinases and phophatases. The present study demonstrates that purified rat brain mitochondria possess endogenous tyrosine phosphatase activity. Mitochondrial phosphatases were found to be capable of dephosphorylating different exogenous substrates, including paranitrophenylphosphate, (32)P-poly(Glu-Tyr)(4:1) and (32)P-angiotensin. These activities are strongly inhibited by peroxovanadate, a well-known inhibitor of tyrosine phosphatases, but not by inhibitors of alkali or Ser/Thr phosphatases, and mainly take place in the intermembrane space and outer mitochondrial membrane. Using a combination of approaches, we identified the tyrosine phosphatase Shp-2 in mitochondria. Shp-2 plays a crucial role in a number of intracellular signalling cascades and is probably involved in several human diseases. It thus represents the first tyrosine phosphatase shown to be present in mitochondria.

Identification and purification from the plasma of Type 1 diabetic subjects of a proteolytically active Grp94Evidence that Grp94 is entirely responsible for plasma proteolytic activity.

AIMS/HYPOTHESIS: The overall increase in proteolytic activity in diabetes is known to be associated with the development and progression of vascular complications. Our aim was to investigate in detail the molecular nature of this activity in the plasma of Type 1 diabetic subjects. METHODS: Plasma of both diabetic and control subjects was subjected to various purification procedures (ion exchange and affinity chromatography, HPLC, immunoprecipitation, electrophoresis, immunoblot and mass analyses) to identify the proteins of interest. Biological activities were measured on specific substrates. RESULTS: In diabetic but not normal plasma we identified the presence of two heat shock proteins, Grp94 (Glucose-regulated protein94) and HSP70. The higher-than-normal proteolytic activity of Grp94 was: (i) directed against casein, but not against endogenous plasma proteins; (ii) fully and specifically inhibited only by anti-Grp94 polyclonal antibodies; and (iii) coupled with low-level ATPase activity. In addition, ATP binding to Grp94 was able to modulate proteolytic activity. We found that Grp94 in plasma circulates only as high molecular mass homo- and hetero-complexes, the latter mostly formed with IgG to which Grp94 is also linked by tenacious binding. Proteolytically-active Grp94 was purified by immunoprecipitation, which co-immunoprecipitated alpha(1)antitrypsin. CONCLUSION/INTERPRETATION: Our results show the unexpected extracellular location and characteristic biological function of Grp94 even at a late stage of disease. These findings have physiopathological relevance for predicting activation of both autoimmune and inflammatory processes potentially associated with vascular complications.

Autocatalytic tyrosine-phosphorylation of protein kinase CK2 alpha and alpha' subunits: implication of Tyr182.

CK2 is a pleiotropic and constitutively active serine/threonine protein kinase composed of two catalytic (alpha and/or alpha') and two regulatory beta-subunits, whose mechanism of modulation is still obscure. Here we show that CK2 alpha/alpha' subunits undergo intermolecular (trans) tyrosine-autophosphorylation, which is dependent on intrinsic catalytic activity and is suppressed by the individual mutation of Tyr182, a crucial residue of the activation loop, to phenylalanine. At variance with serine-autophosphorylation, tyrosine-autophosphorylation of CK2alpha is reversed by ADP and GDP and is counteracted by the beta-subunit and by a peptide reproducing the activation loop of CK2alpha/alpha' (amino acids 175-201). These results disclose new perspectives about the mode of regulation of CK2 catalytic subunits.

Possible implication of the Golgi apparatus casein kinase in the phosphorylation of vesicle docking protein p115 Ser-940: a study with peptide substrates.

Phosphorylation of human vescicle docking protein p115 at Ser-942 (homologous to Ser-940 in rat p115) promotes its dissociation from the Golgi membrane. Here we show that a peptide encompassing the 934--950 sequence of p115 is unaffected or poorly phosphorylated by a variety of Ser/Thr protein kinases with the notable exception of the Golgi apparatus casein kinase (G-CK) which phosphorylates it with an efficiency comparable to that of its optimal peptide substrates. In contrast phosphorylation of the p115 peptide by protein kinase CK2 is negligible compared to that of the specific peptide substrates of this kinase. Phosphorylation by G-CK is abolished if a conserved cluster of acidic residues at position between n + 4 and n + 9 (EDDDDE) is replaced by a neutral stretch (GAGAGA). These data strongly support the view that G-CK but not the other two classes of ubiquitous "casein kinases" (CK1 and CK2) is the natural phosphorylating agent of p115.

Novel consensus sequence for the Golgi apparatus casein kinase, revealed using proline-rich protein-1 (PRP1)-derived peptide substrates.

Previous studies have shown that the Golgi apparatus casein kinase (G-CK) recognizes phosphoacceptor sites specified by the triplet SXE/Sp, which is found in several phosphoproteins, besides casein itself. In the present study, we report that G-CK can phosphorylate, with comparable efficiency, sequences surrounding Ser-22 of salivary proline-rich protein-1 (PRP1), which do not conform to the SXE/Sp motif. By using a series of peptide substrates derived from the PRP1 Ser-22 site, we also have shown that the optimal consensus sequence recognized by G-CK in this case was SXQXX(D/E)3, where the acidic residues at positions n+5 to n+7 and, to a lesser extent, the glutamine residue at position n+2 are the critical determinants.

Sequential phosphorylation of protein band 3 by Syk and Lyn tyrosine kinases in intact human erythrocytes: identification of primary and secondary phosphorylation sites.

Treatment of intact human erythrocytes with pervanadate induces Tyr (Y)-phosphorylation of the transmembrane protein band 3; in parallel, the activity of the immunoprecipitated tyrosine kinases Syk and Lyn is increased. When erythrocytes are incubated with pervanadate together with PP1, a specific inhibitor of Src kinases, including Lyn, the Y-phosphorylation of band 3 is only partially reduced. Indeed, the PP1-resistant phosphorylation of band 3 precedes and is a prerequisite for its coimmunoprecipitation with Lyn, which interacts with the phosphoprotein via the SH2 domain of the enzyme, as proven by binding competition experiments. Upon recruitment to primarily phosphorylated band 3, Lyn catalyzes the secondary phosphorylation of the transmembrane protein. These data are consistent with the view that band 3 is phosphorylated in intact erythrocytes by both PP1-resistant (most likely Syk) and PP1-inhibited (most likely Lyn) tyrosine kinases according to a sequential phosphorylation process. Similar radiolabeled peptide maps are obtained by tryptic digestion of (32)P-band 3 isolated from either pervanadate-treated erythrocytes or red cell membranes incubated with exogenous Syk and Lyn. It has also been demonstrated by means of mass spectrometry that the primary phosphorylation of band 3 occurs at Y8 and Y21, while the secondary phosphorylation affects Y359 and Y904. (Blood. 2000;96:1550-1557)

Ser/Thr phosphorylation of hematopoietic specific protein 1 (HS1): implication of protein kinase CK2.

Hematopoietic lineage cell-specific protein 1 (HS1), a tyrosine multiphosphorylated protein implicated in receptor-mediated apoptosis and proliferative responses, is shown here to become Ser/Thr phosphorylated upon incubation of platelets with radiolabeled inorganic phosphate. The in vivo Ser/Thr phosphorylation of HS1 is enhanced by okadaic acid and reduced by specific inhibitors of casein kinase (CK)2. In vitro, HS1 is an excellent substrate for either CK2 alpha subunit alone (Km = 47 nM) or CK2 holoenzyme, tested in the presence of polylysine (Km = 400 nM). Phosphorylation reaches a stoichiometry of about 2 mol phosphate per mol HS1 and occurs mainly at threonyl residue(s), mostly located in the N-terminal region, but also at seryl residue(s) residing in the central core of the molecule (208-402), as judged from experiments with deleted forms of HS1. Ser/Thr phosphorylation of HS1, either induced in vivo by okadaic acid or catalysed in vitro by CK2, potentiates subsequent phosphorylation at tyrosyl residues. These data indicate the possibility that regulation of HS1 may also be under the control of Ser/Thr phosphorylation, and suggest that in quiescent cells CK2 could play a role in inducing constitutive Tyr phosphorylation of HS1 in the absence of stimuli that activate the protein tyrosine kinase pathway.

GRP94 (endoplasmin) co-purifies with and is phosphorylated by Golgi apparatus casein kinase.

A phosphorylatable protein band of about 94 kDa (as judged by SDS-PAGE) which co-purifies and co-immunoprecipitates with Golgi apparatus casein kinase (G-CK) from rat lactating mammary gland has been shown by mass spectrometric sequence analysis to be identical or very similar to the glucose-regulated protein, GRP94. GRP94 is also readily phosphorylated by G-CK (K(m)=0.2 microM) at seryl sites which are different from the sites affected by casein kinase-2 (CK2) in the same protein. A study with peptide substrates would indicate that the G-CK sites in GRP94 conform to the motif S-R/K-E-X (X being different from D and E) which is not recognized by CK2.

Specific modulatory effect of arachidonic acid on human red blood cell oxalate transport: clinical implications in calcium oxalate nephrolithiasis.

Greater arachidonic acid (AA) contents, which were correlated with erythrocyte transmembrane oxalate (Ox) transport, were observed in plasma and erythrocyte membrane phospholipids of patients with idiopathic calcium renal stones, suggesting a link between membrane phospholipid fatty acid composition and cellular Ox transport. To confirm this hypothesis, the effects of exogenous red blood cell incorporation of three different fatty acids (i.e., oleic acid, AA, and eicosapentaenoic acid) on Ox transport and the phosphorylation status of band 3 protein, which has been shown to mediate red blood cell Ox flux, were investigated. Preincubation of erythrocytes with AA induced a dose-dependent increase in the phosphorylation level of band 3 protein and an increase in transmembrane Ox self-exchange. In contrast, inhibitory effects on both parameters were observed after the incorporation of oleic and eicosapentaenoic acids. These data, together with previous observations of dietary effects on erythrocyte Ox transport and urinary Ox excretion, indicate that genetic and/or nutritional changes in membrane phospholipid fatty acid composition play a crucial role in modulating cellular Ox transport in idiopathic calcium Ox nephrolithiasis.

Hematopoietic lineage cell specific protein 1 associates with and down-regulates protein kinase CK2.

The catalytic (alpha) subunit of protein kinase CK2 and the hematopoietic specific protein 1 (HS1) display opposite effects on Ha-ras induced fibroblast transformation, by enhancing and counteracting it, respectively. Here we show the occurrence of physical association between HS1 and CK2alpha as judged from both far Western blot and plasmon resonance (BIAcore) analysis. Association of HS1 with CK2alpha is drastically reduced by the deletion of the HS1 C-terminal region (403-486) containing an SH3 domain. HS1, but not its deletion mutant HS1 Delta324-393, lacking a sequence similar to an acidic stretch of the regulatory beta-subunit of CK2, inhibits calmodulin phosphorylation by CK2alpha. These data indicate that HS1 physically interacts with CK2alpha and down-regulates its activity by a mechanism similar to the beta-subunit.

Molecular features underlying the sequential phosphorylation of HS1 protein and its association with c-Fgr protein-tyrosine kinase.

The hematopoietic lineage cell-specific protein HS1 was shown to undergo a process of sequential phosphorylation both in vitro and in vivo, which is synergistically mediated by Syk and Src family protein-tyrosine kinases and essential for B cell antigen receptor-mediated apoptosis. We have now identified tyrosine 222 as the HS1 residue phosphorylated by the Src family protein kinases c-Fgr and Lyn, and we show that a truncated form of HS1 (HS1-208-401) lacking the N-terminal putative DNA binding region and the C-terminal Src homology 3 (SH3) domain is still able to undergo all the steps of sequential phosphorylation as efficiently as full-length HS1. We also show that a stable association of phospho-HS1 with c-Fgr through its SH2 domain requires previous autophosphorylation of the kinase and is prevented by subsequent phosphorylation of Tyr-222. Kinetic studies with HS1 and its truncated forms previously phosphorylated by Syk and with a peptide substrate reproducing the sequence around tyrosine 222 support the view that efficient phosphorylation of HS1 by Src family protein kinases entirely relies on TyrP-SH2 domain interaction with negligible, if any, contribution of local specificity determinants. Our data indicate that the proline-rich region of HS1 bordered by tyrosyl residues affected by Syk and Src family kinases represents a functional domain designed to undergo a process of sequential phosphorylation.

Src homology-2 domains protect phosphotyrosyl residues against enzymatic dephosphorylation.

The SH2 domain of c-Fgr (class 1A) has been expressed in E. coli as GST fusion protein and tested for its ability to prevent the dephosphorylation of a variety of phosphotyrosyl (poly)peptides by three distinct protein tyrosine phosphatases (TC-PTPase, YOP, and Low Mr PTPase). Dephosphorylation of HS1 protein and of a derived phosphopeptide, HS1 (388-402), exhibiting the motif selected by class 1A SH2 domains is inhibited in a dose dependent manner with full inhibition promoted by a 2- to 3-molar excess of GST/SH2 domain irrespective of either the nature or the amount of phosphatase used. The IC50 values for inhibition of these and other phosphotyrosyl substrates roughly correlates with their expected affinity for class 1A SH2 domain. Inhibition is partially reversed by the addition of D-myo-inositol 1,4,5-triphosphate, which competes for the binding to the SH2 domains. Our data on one side show that additional mechanism(s) besides mere competition must assist PTPases to dissociate SH2-PTyr complexes and on the other suggest a role for SH2 domains in protecting phosphotyrosyl residues from premature dephosphorylation.

Spontaneous autophosphorylation of Lyn tyrosine kinase at both its activation segment and C-terminal tail confers altered substrate specificity.

Two tyrosyl residues have been reported to play a crucial role in the regulation of protein tyrosine kinases of the Src family: autophosphorylation of Tyr416 (c-Src numbering) located in the catalytic domain correlates with enzyme activation, while Csk-mediated phosphorylation of the C-terminal tyrosine Tyr527 (c-Src numbering) gives rise to inactive forms of Src kinases. Here we show that the Src-related Lyn kinase undergoes spontaneous and stoichiometric autophosphorylation at both Tyr396 (homologous to c-Src Tyr416) and Tyr507 (homologous to c-Src Tyr527). Such a doubly autophosphorylated form of Lyn is hyperactive toward peptide substrates and insensitive to Csk-induced downregulation. In contrast, doubly autophosphorylated Lyn exhibits reduced activity toward protein substrates such as phospho-p50/HS1 (hematopoietic-lineage cell-specific protein) and p57/PDI (protein disulfide isomerase related protein), whose multiple sequential/processive phosphorylation relies on the accessibility of the SH2 domain of the kinase. These data disclose a novel conformation of Lyn that is catalytically active despite the presence of an intramolecular interaction between the phosphorylated tail and the SH2 domain. This enzyme conformation is expected to display a reduced oncogenic potential resulting from its defective recognition of a subset of protein substrates whose targeting is mediated by the Lyn SH2 domain.

Sequence specificity of C-terminal Src kinase (CSK)--a comparison with Src-related kinases c-Fgr and Lyn.

An eicosapeptide encompassing the C-terminal tail of c-Src (Tyr527) which is conserved in most Src-related protein kinases, is phosphorylated by C-terminal Src kinase (CSK) and by the two Src-related protein kinases c-Fgr and Lyn, with similar kinetic constants. Two related peptides reproducing the C-terminal segments of c-Src mutants defective in CSK phosphorylation [MacAuley, A., Okada, M., Nada, S., Nakagawa, H. & Cooper, J. A. (1993) Oncogene 8, 117-124] AFLEDSCTGTEPLYQRGENL (mutant number 28) and AFLEDNFTGTKPQYHPGENL (mutant number 29), proved a better and a much worse substrates, respectively than the wild-type peptide, with either CSK or the two Src kinases. By changing individual residues in the best peptide substrate, it was shown that the main element responsible for its improved phosphorylation is leucine at position -1 (instead of glutamine), while lysine at position -3 (instead of glutamate) has a detrimental effect, possibly accounting for the negligible phosphorylation of peptide derived from mutant number 29. By contrast to most peptide substrates, including the Src C-terminal peptides, which exhibit relatively high K(m) values, a polyoma-virus-middle-T-antigen-(mT)-derived peptide with tyrosine embedded in a highly hydrophobic sequence (EEEPQFEEIPIYLELLP) exhibits with CSK a quite low K(m) value (63 microM). Consistent with this, the optimal sequence selected by CSK in an oriented peptide library is XXXIYMFFF. This is different from sequences selected by Lyn (DEEIYEELX) and c-Fgr (XEEIYGIFF), although they all share a high selection for a hydrophobic residue at n-1. In sharp contrast, TPKIIB/p38syk, related to the catalytic domain of p72syk, selects acidic residues at nearly all positions, n-1 included. These data support the notion that the features determining the specific phosphorylation of the C-terminal tyrosine residue of Src do not reside in the primary structure surrounding the target tyrosine. They also show that this site does not entirely fulfil the optimal consensus sequence recognized by CSK, disclosing the possibility that as yet unrecognized CSK targets structurally unrelated to the C-terminal tyrosine residue of Src kinases may exist.

Specific stimulation of c-Fgr kinase by tyrosine-phosphorylated (poly)peptides--possible implication in the sequential mode of protein phosphorylation.

Hematopoietic lineage cell-specific HS1 protein is converted into a substrate for c-Fgr by previous Syk-mediated phosphorylation, at site(s) that bind to the SH2 domain of c-Fgr [Ruzzene, M., Brunati, A. M., Marin, O., Donella-Deana, A. & Pinna, L. A. (1996) Biochemistry 35, 5327-5332]. Here we show that a phosphopeptide derived from one such site, HS1-(320-329)-phosphopeptide (PEGDYpEEVLE), enhances up to tenfold, in a dose-dependent manner, the catalytic activity of c-Fgr either assayed with peptide substrates or evaluated as intermolecular autophosphorylation of c-Fgr itself. The dephosphorylated HS1-(320-329)-peptide is totally ineffective, while the stimulatory efficacy of other phosphopeptides derived from the polyoma virus middle T antigen-(393-402) sequence, c-Src, and c-Fgr autophosphorylation sites, and the C-terminal c-Src site (Tyr527) is variable and correlates reasonably well with the predicted affinity for the c-Fgr SH2 domain. Stimulation of c-Fgr catalytic activity is also promoted by the full-length HS1 protein, previously tyrosine phosphorylated by Syk, and is accounted for by an increased Vmax while the Km values are unchanged. If the normal activator of c-Fgr kinase, Mg2+, is replaced by Mn2+, stimulation by HS1-(320-329)-phosphopeptide is still observable with peptide substrates, while autophosphorylation is, in contrast, inhibited by the phosphopeptide. These findings, in conjunction with the ability of previously autophosphorylated c-Fgr to be stimulated by HS1-(320-329)-phosphopeptide, support the view that stimulation of c-Fgr by phosphopeptide is not or is not entirely a consequence of increased autophosphorylation. Interestingly, neither Syk and C-terminal Src kinase nor three other members of the Src family (Lyn, Lck, and Fyn) are susceptible to stimulation by phosphopeptide, as observed with c-Fgr. These data support the notion that c-Fgr undergoes a unique mechanism of activation promoted by tyrosine-phosphorylated polypeptide that binds to its SH2 domain. This suggests that such a mode of regulation is peculiar of protein-tyrosine kinases committed to the secondary phosphorylation of sequentially phosphorylated proteins.