Intestinal crypt stem cells possess high levels of cytoskeletal- associated phosphotyrosine-containing proteins and tyrosine kinase activity relative to differentiated enterocytes

Growth and differentiation of stem cells is thought to be regulated by growth factors and responding protein tyrosine kinase activities. Comparing mitotic stem cells from the adult intestinal epithelium, isolated from the crypts of Lieberkuhn, with isolated differentiated absorbtive cells we find major differences in the levels of phosphotyrosine-containing proteins. Crypt stem cells possess two major phosphotyrosine-containing polypeptides of 36 and 17 kD which have greater than 15 times more phosphotyrosine than that present in the polypeptides of differentiated enterocytes. Tyrosine kinase activity and similar phosphotyrosine-containing proteins are associated with the Triton cytoskeleton. Moreover, crypt tyrosine kinase(s) is active in vitro in phosphorylating similar cytoskeleton-associated substrates. These results suggest that cytoskeleton-associated phosphotyrosine kinase(s) and their substrates may play a role in growth and differentiation of adult intestinal epithelial cells.     

Characterization of kinase inhibitors using different phosphorylation states of colony stimulating factor-1 receptor tyrosine kinase

It is known that some kinase inhibitors are sensitive to the phosphorylation state of the kinase, and therefore those compounds can discriminate between a phosphorylated and unphosphorylated protein. In this study, we prepared two colony stimulating factor-1 receptor (CSF-1R) tyrosine kinase proteins: one highly phosphorylated by autophosphorylation and the other dephosphorylated by phosphatase treatment. These kinases were subjected to an activity-based assay to investigate the effect of their phosphorylation state on the potency of several kinase inhibitors. Dasatinib, sorafenib, PD173074 and staurosporine showed similar inhibition against different phosphorylation states of CSF-1R, but pazopanib, sunitinib, GW2580 and imatinib showed more potent inhibition against dephosphorylated CSF-1R. Binding analysis of the inhibitors to the two different phosphorylation forms of CSF-1R, using surface plasmon resonance spectrometry, revealed that staurosporine bound to both forms with similar affinity, but sunitinib bound to the dephosphorylated form with higher affinity. Thus, these observations suggest that sunitinib binds preferentially to the inactive form, preventing the activation of CSF-1R. Screening against different activation states of kinases should be an important approach for prioritizing compounds and should facilitate inhibitor design.     

Epidermal growth factor stimulates tyrosine phosphorylation of specific proteins in permeabilized human fibroblasts

We have investigated the epidermal growth factor (EGF)-stimulated tyrosine-specific protein kinase activity in quiescent cultures of diploid human fibroblasts that have a well characterized mitogenic response to EGF. We developed a method of permeabilizing cells with digitonin or other agents that permitted the rapid labeling of cellular proteins with exogenously added [gamma-32P]ATP while allowing only about 25% of marker cytosolic enzymes to escape from the cells. When phosphatases were inhibited with zinc and vanadate, EGF induced up to 8-fold stimulation of the incorporation of radioactivity from [gamma-32P]ATP into a 35-kDa band on sodium dodecyl sulfate gels. Alkali treatment of gels showed that EGF stimulated the phosphorylation of bands with apparent molecular masses of 170, 45, 35, 26, 22, and 21 kDa. Phosphoamino acid analysis was performed on the 170- and 35-kDa bands and revealed that the EGF-stimulated phosphorylation was on tyrosyl residues. The 35-kDa band was resolved into four spots by two-dimensional gel electrophoresis. The most acidic form was the most prominent and it was precipitated by an antiserum against a 35-kDa protein from A-431 cells; heretofore, this protein has only been reported to be phosphorylated in an EGF-dependent manner by A-431 membranes in vitro (Fava, R. A., and Cohen, S. (1984) J. Biol. Chem. 259, 2636-2645). This antiserum also precipitated a 35-kDa phospho-protein from extracts of intact [32P]orthophosphate-labeled fibroblasts which was phosphorylated on tyrosine in an EGF-dependent manner. None of the forms of the 35-kDa phosphoproteins labeled in permeabilized cells were immunologically related to the 34-kDa protein that is a substrate for the tyrosyl kinase encoded by Rous sarcoma virus. Other mitogens (serum, insulin, platelet-derived growth factor, and thrombin) did not detectably stimulate phosphorylation in permeabilized cells.     

Cytosolic and stored calcium antagonistically control tyrosine phosphorylation of specific platelet proteins.

Depletion of intracellular calcium stores appears to increase plasma membrane permeability for calcium by an as yet obscure mechanism. We found that the Ca2+ ionophore, A23187, and thrombin elevate cytosolic calcium ([Ca2+]i) equally and cause tyrosine phosphorylation of a 130-kDa protein and to a lesser extent 80- and 60-kDa proteins. Chelation of [Ca2+]i by 1,2-bis(2-aminophenoxyethane)-N,N,N',N'-tetraacetic acid/acetomethoxy ester decreased thrombin-induced tyrosine phosphorylation responses. These results suggested that [Ca2+]i elevation promotes tyrosine phosphorylation. Tyrosine phosphorylation persisted in the presence or absence of extracellular calcium after thrombin stimulation but subsided rapidly after A23187 addition if extracellular calcium was present. When Ca2+/ATPase activity, which is apparently required to maintain calcium stores, is inhibited by low temperature, tyrosine phosphorylation of the 130-kDa protein occurs. Rewarming platelets reverses tyrosine phosphorylation only if extracellular calcium is present. Thapsigargin, a calcium ATPase inhibitor, also induces tyrosine phosphorylation of the 130-kDa protein and prevents dephosphorylation of this protein when added prior to rewarming. These observations suggest that homeostatic levels of calcium in storage compartments favor tyrosine dephosphorylation of specific proteins. Thus the levels of [Ca2+]i and stored calcium appear to control tyrosine phosphorylation antagonistically. Tyrosine phosphorylation may play a role in regulating calcium channel function.     

Negative regulation of receptor tyrosine kinase signals

In Metazoans a number of cellular functions are controlled by receptor tyrosine kinases (RTKs) during development and in postnatal life. The execution of these programs requires that signals of adequate strength are delivered for the appropriate time within precise spatial boundaries. Several RTK inhibitors have been identified in invertebrate and mammalian organisms. Because they are involved in tuning and termination of receptor signals, negative regulators of RTK activity fulfill a fundamental function in the control of receptor signaling.     

Conserved Cdk inhibitors show unique structural responses to tyrosine phosphorylation

Balanced proliferation-quiescence decisions are vital during normal development and in tissue homeostasis, and their dysregulation underlies tumorigenesis. Entry into proliferative cycles is driven by Cyclin/Cyclin-dependent kinases (Cdks). Conserved Cdk inhibitors (CKIs) p21^Cip1/Waf1, p27^Kip1, and p57^Kip2 bind to Cyclin/Cdks and inhibit Cdk activity. p27 tyrosine phosphorylation, in response to mitogenic signaling, promotes activation of CyclinD/Cdk4 and CyclinA/Cdk2. Tyrosine phosphorylation is conserved in p21 and p57, although the number of sites differs. We use molecular-dynamics simulations to compare the structural changes in Cyclin/Cdk/CKI trimers induced by single and multiple tyrosine phosphorylation in CKIs and their impact on CyclinD/Cdk4 and CyclinA/Cdk2 activity. Despite shared structural features, CKI binding induces distinct structural responses in Cyclin/Cdks and the predicted effects of CKI tyrosine phosphorylation on Cdk activity are not conserved across CKIs. Our analyses suggest how CKIs may have evolved to be sensitive to different inputs to give context-dependent control of Cdk activity.     

Adherence of platelets under flow conditions results in specific phosphorylation of proteins at tyrosine residues

Collagen is a powerful platelet activating agent that promotes adhesion and aggregation of platelets. To differentiate the signals generated in these processes we have analyzed the tyrosine phosphorylation occurring in platelets after activation with collagen in suspension or under flow conditions. For the suspension studies, washed platelets were activated with different concentrations of purified type I collagen (ColI). Studies under flow conditions were performed using two different adhesive substrata: ColI and endothelial cells extracellular matrix (ECM). Coverslips coated with ColI or ECM were perfused through a parallel-plate perfusion chamber at 800 s(-1) for 5 min. After activation of platelets either in suspension or by adhesion, samples were solubilized and proteins were resolved by electrophoresis. Tyrosine-phosphorylated proteins were detected in immunoblots by specific antibodies. Activation of platelet suspensions with collagen induced tyrosine phosphorylation before aggregation could be detected. Profiles showing tyrosine-phosphorylated proteins from platelets adhered on ColI or on ECM were almost identical and lacked proteins p95, p80, p66, and p64, which were present in profiles from platelets activated in suspension. The intensity of phosphorylation was quantitatively weaker in those profiles from platelets adhered on ECM. Results from the present work indicate that activation of platelets in suspension or by adhesion induces differential tyrosine phosphorylation patterns. Phosphorylation of proteins p90 and p76 may be related to early activation events occurring during initial contact and spreading of platelets. Considering that adhesion is the first step of platelet activation, studies on signal transduction mechanisms under flow conditions may provide new insights to understand the signaling processes taking place at earliest stages of platelet activation.     

Acquired resistance to tyrosine kinase inhibitors during cancer therapy

Selective tyrosine kinase inhibitors have emerged as important therapeutic agents in the treatment of a variety of human malignancies. Although several of these inhibitors have marked clinical activity, it is widely recognized that the overall value of these agents is substantially limited by the acquisition of drug resistance, which eventually arises in most, if not all treated patients. Mechanisms of drug resistance are beginning to be elucidated through the molecular analysis of clinical specimens as well as through cell culture modeling. By identifying resistance mechanisms, it should be possible to develop 'second-generation' inhibitors as well as rational drug combinations that can overcome or even prevent acquired resistance to kinase inhibitors, thereby enhancing clinical benefit.     

Development of N-4,6-pyrimidine-N-alkyl-N'-phenyl ureas as orally active inhibitors of lymphocyte specific tyrosine kinase

A new class of lymphocyte specific tyrosine kinase (lck) inhibitors based on an N-4,6-pyrimidine-N-alkyl-N'-phenyl urea scaffold is described. Many of these compounds showed low-nanomolar inhibition of lck kinase activity as well as IL-2 synthesis from Jurkat cells. One of these analogs, 7i, was shown to be orally efficacious by in vivo testing in a rat adjuvant-induced arthritis study.     

Mitogen-stimulated phosphorylation of nuclear proteins in Swiss 3T3 cells: evidence for a protein kinase C requirement

When Swiss 3T3 fibroblasts are treated with a combination of IGF-I2 and bombesin at mitogenic concentrations, in vivo phosphorylation of some nuclear proteins occurs within 45-90 min. Among these proteins, histone H1 and a 0.75 M PCA soluble polypeptide with an apparent Mr of 21,000, as revealed by electrophoretic analysis, are phosphorylated in vitro by protein kinase C in isolated nuclei purified from 3T3 cells treated for 90 min with IGF-I and bombesin. Since these phosphorylative events follow the earlier changes, recently demonstrated, in nuclear polyphosphoinositide metabolism induced by the same mitogen combination, it seems possible that these two phenomena are related to each other and trigger the synthetic machinery responsible for replicating DNA.     

Tailoring tyrosine kinase inhibitors to fit the lung cancer genome

Tyrosine kinase inhibitors (TKIs) have been in use as cancer therapeutics for nearly a decade, and their utility in targeting specific malignancies with defined genetic lesions has proven to be remarkably effective. Recent efforts to characterize the spectrum of genetic lesions found in non-small cell lung carcinoma (NSCLC) have provided important insights into the molecular basis of this disease and have also revealed a wide array of tyrosine kinases that might be effectively targeted for rationally designed therapies. The findings of these studies, however, also provide a cautionary tale about the limitations of single-agent therapies, which fail to account for the genetic heterogeneity and pathway redundancy that characterize advanced NSCLC. Emergence of drug resistance mechanisms to specific TKIs, such as gefitinib and erlotinib, suggests that more sophisticated chemotherapeutic paradigms that target multiple pathways at the same time will be required to effectively treat this disease.     

Urokinase receptor expression involves tyrosine phosphorylation of phosphoglycerate kinase

The interaction of urokinase-type plasminogen activator (uPA) with its receptor, uPAR, plays a central role in several pathophysiological processes, including cancer. uPA induces its own cell surface receptor expression through stabilization of uPAR mRNA. The mechanism involves binding of a 51 nt uPAR mRNA coding sequence with phosphoglycerate kinase (PGK) to down regulate cell surface uPAR expression. Tyrosine phosphorylation of PGK mediated by uPA treatment enhances uPAR mRNA stabilization. In contrast, inhibition of tyrosine phosphorylation augments PGK binding to uPAR mRNA and attenuates uPA-induced uPAR expression. Mapping the specific peptide region of PGK indicated that its first quarter (amino acids 1–100) interacts with uPAR mRNA. To determine if uPAR expression by uPA is regulated through activation of tyrosine residues of PGK, we mutated the specific tyrosine residue and tested mutant PGK for its ability to interfere with uPAR expression. Inhibition of tyrosine phosphorylation by mutating Y76 residue abolished uPAR expression induced by uPA treatment. These findings collectively demonstrate that Y76 residue present in the first quarter of the PGK molecule is involved in lung epithelial cell surface uPAR expression. This region can effectively mimic the function of a whole PGK molecule in inhibiting tumor cell growth.     

Mechanism of action of erbB tyrosine kinase inhibitors

Over the last decade, drug discovery efforts have generated a myriad of compounds that inhibit the activity of the erbB family of tyrosine kinases with potencies and selectivity that have surpassed original expectations. These characteristics, along with improved pharmaceutical properties, have enabled inhibitors from this class of agents to finally realize their therapeutic potential, and indeed, some are currently producing significant clinical responses. Interestingly, those properties that are essential for a clinically active inhibitor of the erbB family are most readily attained with compounds that bind at the ATP site, and the most successful compounds have shown a distinct convergence to certain common chemical features. The reasons for this trend are beginning to be realized through the generation of an increasing array of crystalline structures for protein kinases as well as advances in molecular modeling. This has allowed a more complete understanding of the precise physical interactions that occur between erbB tyrosine kinase inhibitors and their target(s), which, in turn, has begun to shed light on the mechanism by which these molecules attain their remarkable affinity and specificity.     

Inhibition of Bcr serine kinase by tyrosine phosphorylation.

The first exon of the BCR gene encodes a new serine/threonine protein kinase. Abnormal fusion of the BCR and ABL genes, resulting from the formation of the Philadelphia chromosome (Ph), is the hallmark of Ph-positive leukemia. We have previously demonstrated that the Bcr protein is tyrosine phosphorylated within first-exon sequences by the Bcr-Abl oncoprotein. Here we report that in addition to tyrose 177 (Y-177), Y-360 and Y283 are phosphorylated in Bcr-Abl proteins in vitro. Moreover, Bcr tyrosine 360 is phosphorylated in vivo within both Bcr-Abl and Bcr. Bcr mutant Y177F had a greatly reduced ability to transphosphorylate casein and histone H1, whereas Bcr mutants Y177F and Y283F had wild-type activities. In contrast, the Y360F mutation had little effect on Bcr's autophosphorylation activity. Tyrosine-phosphorylated Bcr, phosphorylated in vitro by Bcr-Abl, was greatly inhibited in its serine/threonine kinase activity, impairing both auto- and transkinase activities of Bcr. Similarly, the isolation of Bcr from cells expressing Bcr-Abl under conditions that preserve phosphotyrosine residues also reduced Bcr's kinase activity. These results indicate that tyrosine 360 of Bcr is critical for the transphosphorylation activity of Bcr and that in Ph-positive leukemia, Bcr serine/threonine kinase activity is seriously impaired.     

Characterization of fodrin phosphorylation by spleen protein tyrosine kinase

Fodrin, an actin and calmodulin binding and spectrin-like protein present in many nonerythrocyte tissues, could be phosphorylated up to more than 1.5 mol of phosphate/mol of protein by a highly purified non-receptor-associated protein tyrosine kinase from bovine spleen. The protein phosphorylation was not affected by Ca2+/calmodulin or by F-actin. Km and Vmax values of the reaction were 91 nM and 0.35 nmol of P2 min-1 (mg of kinase)-1, respectively. Both subunits A and B of fodrin were phosphorylated, with the rate of subunit A phosphorylation much greater than that of subunit B phosphorylation. Tryptic phosphopeptide mapping of the phosphorylated subunits suggested that there were three major phosphorylation sites in subunit A and one in subunit B. Phosphotyrosylfodrin could be dephosphorylated by the calmodulin-stimulated phosphatase (calcineurin) in the presence of activating metal ions; Ni2+ was a much more effective activator than Mn2+ for this reaction. Fodrin phosphorylation by the spleen protein tyrosine kinase did not appear to alter the actin and calmodulin binding properties of the protein. On the other hand, the calmodulin-dependent stimulation of smooth muscle actomyosin Mg2+-ATPase by fodrin was enhanced by 101% +/- 3% (n = 3) upon fodrin phosphorylation. Ni2+-calcineurin, which was shown to effectively dephosphorylate the phosphotyrosyl residues on fodrin, could reverse the phosphorylation-enhanced Mg2+-ATPase stimulatory activity of fodrin.     

Substrate phosphorylation catalyzed by the insulin receptor tyrosine kinase. Kinetic correlation to autophosphorylation of specific sites in the beta subunit

The kinetics of insulin-stimulated autophosphorylation of specific tyrosines in the beta subunit of the mouse insulin receptor and activation of receptor kinase-catalyzed phosphorylation of a model substrate were compared. The deduced amino acid sequence of the mouse proreceptor was determined to locate tyrosine-containing tryptic peptides. Receptor was first incubated with unlabeled ATP to occupy nonrelevant autophosphorylation sites, after which [32P]autophosphorylation at relevant sites and attendant activation of substrate phosphorylation were initiated with [gamma-32P]ATP and insulin. Activation of substrate phosphorylation underwent an initial lag of 10-20 s during which there was substantial 32P-autophosphorylation of tryptic phosphopeptides p2 and p3, but not p1. Following the lag, incorporation of 32P into p1 and the activation of substrate phosphorylation increased abruptly and exhibited identical kinetics. The addition of substrate to the receptor prior to ATP inhibits insulin-stimulated autophosphorylation, and consequently substrate phosphorylation. Insulin-stimulated autophosphorylation of the receptor in the presence of substrate inhibited primarily the incorporation of 32P into p1 and drastically inhibited substrate phosphorylation. From Edman radiosequencing of 32P-labeled p1, p2, and p3 and the amino acid sequence of the mouse receptor, the location of each phosphopeptide within the beta subunit was determined. Further characterization of these phosphopeptides revealed that p1 and p2 represent the triply and doubly phosphorylated forms, respectively, of the region within the tyrosine kinase domain containing tyrosines 1148, 1152, and 1153. The doubly phosphorylated forms contain phosphotyrosines either at positions 1148 and 1152/1153 or positions 1152 and 1153. These results indicate that insulin stimulates sequential autophosphorylation of tyrosines 1148, 1152 and 1153, and that the transition from the doubly to the triply phosphorylated forms is primarily responsible for the activation of substrate phosphorylation.     

Endocytosis of Candida albicans by vascular endothelial cells is associated with tyrosine phosphorylation of specific host cell proteins

Candida albicans escapes from the bloodstream by invading the endothelial cell lining of the vasculature. In vitro, C. albicans invades endothelial cells by inducing its own endocytosis. We examined whether this process is regulated by the tyrosine phosphorylation of endothelial cell proteins. We found that endocytosis of wild-type C. albicans was accompanied by the tyrosine phosphorylation of two endothelial cell proteins with molecular masses of 80 and 82 kDa. The phosphorylation of these proteins was closely associated with the endocytosis of C. albicans because these proteins were phosphorylated in response to the endocytosis of both live and killed organisms, but they were not phosphorylated in endothelial cells infected with a poorly endocytosed strain of C. albicans. The tyrosine kinase inhibitors genistein and tyrphostin 47 blocked the phosphorylation of the two endothelial cell proteins and significantly reduced endocytosis of C. albicans. Therefore, C. albicans probably induces its own endocytosis by stimulating the tyrosine phosphorylation of two endothelial cell proteins.