Lars Rönnstrand

Signal transduction via platelet-derived growth factor receptors

Platelet-derived growth factor (PDGF) exerts its stimulatory effects on cell growth and motility by binding to two related protein tyrosine kinase receptors. Ligand binding induces receptor dimerization and autophosphorylation, allowing binding and activation of cytoplasmic SH2-domain containing signal transduction molecules. Thereby, a number of different signaling pathways are initiated leading to cell growth, actin reorganization migration and differentiation. Recent observations suggest that extensive cross-talk occurs between different signaling pathways, and that stimulatory signals are modulated by inhibitory signals arising in parallel.

Chk1 regulates the S phase checkpoint by coupling the physiological turnover and ionizing radiation-induced accelerated proteolysis of Cdc25A

Chk1 kinase coordinates cell cycle progression and preserves genome integrity. Here, we show that chemical or genetic ablation of human Chk1 triggered supraphysiological accumulation of the S phase-promoting Cdc25A phosphatase, prevented ionizing radiation (IR)-induced degradation of Cdc25A, and caused radioresistant DNA synthesis (RDS). The basal turnover of Cdc25A operating in unperturbed S phase required Chk1-dependent phosphorylation of serines 123, 178, 278, and 292. IR-induced acceleration of Cdc25A proteolysis correlated with increased phosphate incorporation into these residues generated by a combined action of Chk1 and Chk2 kinases. Finally, phosphorylation of Chk1 by ATM was required to fully accelerate the IR-induced degradation of Cdc25A. Our results provide evidence that the mammalian S phase checkpoint functions via amplification of physiologically operating, Chk1-dependent mechanisms.

Binding of different dimeric forms of PDGF to human fibroblasts: evidence for two separate receptor types

The binding of the three dimeric forms of platelet‐derived growth factor (PDGF), PDGF‐AA, PDGF‐AB and PDGF‐BB, to human fibroblasts was studied. Cross‐competition experiments revealed the existence of two different PDGF receptor classes: the type A PDGF receptor bound all three dimeric forms of PDGF, whereas the type B PDGF receptor bound PDGF‐BB with high affinity and PDGF‐AB with lower affinity, but not PDGF‐AA. The sizes of the two receptors were estimated with affinity labeling techniques; the A type receptor appeared as a major component of 125 kd and a minor of 160 kd, and the B type receptor as two components of 160 and 175 kd. A previously established PDGF receptor monoclonal antibody, PDGFR‐B2, was shown to react with the B type receptor only. The different abilities of the three dimeric forms of PDGF to stimulate incorporation of [3H]TdR into human fibroblasts indicated that the major mitogenic effect of PDGF is mediated via the B type receptor.

Identification of Novel Phosphorylation Sites in Hormone-sensitive Lipase That Are Phosphorylated in Response to Isoproterenol and Govern Activation Properties in Vitro

Hormone-sensitive lipase (HSL) is the rate-limiting enzyme in lipolysis. Stimulation of rat adipocytes with isoproterenol results in phosphorylation of HSL and a 50-fold increase in the rate of lipolysis. In this study, we used site-directed mutagenesis and two-dimensional phosphopeptide mapping to show that phosphorylation sites other than the previously identified Ser-563 are phosphorylated in HSL in response to isoproterenol stimulation of32P-labeled rat adipocytes. Phosphorylation of HSL in adipocytes in response to isoproterenol and in vitrophosphorylation of HSL containing Ser → Ala mutations in residues 563 and 565 (S563A,S565A) with protein kinase A (PKA), followed by tryptic phosphopeptide mapping resulted in two tryptic phosphopeptides. These tryptic phosphopeptides co-migrated with the phosphopeptides released by the same treatment of F654HPRRSSQGVLHMPLYSSPIVK675phosphorylated with PKA. Analysis of the phosphorylation site mutants, S659A, S660A, and S659A,S660A disclosed that mutagenesis of both Ser-659 and Ser-660 was necessary to abolish the activation of HSL toward a triolein substrate after phosphorylation with PKA. Mutation of Ser-563 to alanine did not cause significant change of activation compared with wild-type HSL. Hence, our results demonstrate that in addition to the previously identified Ser-563, two other PKA phosphorylation sites, Ser-659 and Ser-660, are present in HSL and, furthermore, that Ser-659 and Ser-660 are the major activity controlling sites in vitro.

Signal transduction via the stem cell factor receptor/c-Kit.

Abstract.Together with its ligand, stem cell factor, the receptor tyrosine kinase c-Kit is a key controlling receptor for a number of cell types, including hematopoietic stem cells, mast cells, melanocytes and germ cells. Gain-of-function mutations in c-Kit have been described in a number of human cancers, including testicular germinomas, acute myeloid leukemia and gastrointestinal stromal tumors.Stimulation of c-Kit by its ligand leads to dimerization of receptors, activation of its intrinsic tyrosine kinase activity and phosphorylation of key tyrosine residues within the receptor. These phosphorylated tyrosine residues serve as docking sites for a number of signal transduction molecules containing Src homology 2 domains, which will thereby be recruited to the receptor and activated many times through phosphorylation by the receptor. This review discusses our current knowledge of signal transduction molecules and signal transduction pathways activated by c-Kit and how their activation can be connected to the physiological outcome of c-Kit signaling.

Stem Cell Factor Receptor/c-Kit: From Basic Science to Clinical Implications

Stem cell factor (SCF) is a dimeric molecule that exerts its biological functions by binding to and activating the receptor tyrosine kinase c-Kit. Activation of c-Kit leads to its autophosphorylation and initiation of signal transduction. Signaling proteins are recruited to activated c-Kit by certain interaction domains (e.g., SH2 and PTB) that specifically bind to phosphorylated tyrosine residues in the intracellular region of c-Kit. Activation of c-Kit signaling has been found to mediate cell survival, migration, and proliferation depending on the cell type. Signaling from c-Kit is crucial for normal hematopoiesis, pigmentation, fertility, gut movement, and some aspects of the nervous system. Deregulated c-Kit kinase activity has been found in a number of pathological conditions, including cancer and allergy. The observation that gain-of-function mutations in c-Kit can promote tumor formation and progression has stimulated the development of therapeutics agents targeting this receptor, e.g., the clinically used inhibitor imatinib mesylate. Also other clinically used multiselective kinase inhibitors, for instance, sorafenib and sunitinib, have c-Kit included in their range of targets. Furthermore, loss-of-function mutations in c-Kit have been observed and shown to give rise to a condition called piebaldism. This review provides a summary of our current knowledge regarding structural and functional aspects of c-Kit signaling both under normal and pathological conditions, as well as advances in the development of low-molecular-weight molecules inhibiting c-Kit function.

Identification of two juxtamembrane autophosphorylation sites in the PDGF beta-receptor; involvement in the interaction with Src family tyrosine kinases.

Two novel sites of autophosphorylation were localized to the juxtamembrane segment of the human platelet‐derived growth factor (PDGF) beta‐receptor. To evaluate the importance of these phosphorylation sites, receptor mutants were made in which Tyr579, Tyr581 or both were replaced with phenylalanine residues; the receptor mutants were stably expressed in porcine aortic endothelial cells. Compared with the wild‐type receptor, the Y579F and Y581F mutants were less able to mediate association with and activation of the Src family tyrosine kinases. The ability of these phosphorylation sites to mediate directly the binding of the Src family proteins was also demonstrated by using phosphotyrosine‐containing synthetic peptides representing the juxtamembrane sequence of the receptor. Both the Y579F and Y581F mutants were similar to the wild‐type receptor with regard to their protein tyrosine kinase activity and ability to induce mitogenicity in response to PDGF‐BB. A conclusive evaluation of the role of the Src family members in signal transduction could, however, not be made since our attempt to prevent completely the association by mutation of both Tyr579 and Tyr581, resulted in loss of kinase activity and was therefore not informative. The present data, together with previous observations, demonstrate a high degree of specificity in the interaction between different autophosphorylation sites in the PDGF beta‐receptor and downstream components in the signal transduction pathway.

INDUCTION OF B-TYPE RECEPTORS FOR PLATELET-DERIVED GROWTH FACTOR IN VASCULAR INFLAMMATION: POSSIBLE IMPLICATIONS FOR DEVELOPMENT OF VASCULAR PROLIFERATIVE LESIONS

Expression of B-type receptors for platelet-derived growth factor (PDGF) in frozen sections of blood vessels from tissues affected by abnormal vascular cell proliferation was investigated by immunohistochemical techniques and compared with expression of these receptors in blood vessels of normal tissues. Receptors were not expressed, or expressed at low levels, in vessels of normal tissues. In contrast, a pronounced expression of PDGF B-type receptors was seen on vascular smooth muscle cells in atherosclerotic plaques, rejected kidneys, and chronic synovitis. These observations suggest induction of PDGF B-type receptors on vascular smooth muscle cells in inflamed tissues, which would render such cells responsive to growth stimulation by PDGF released from captured platelets, or produced locally (eg, by inflammatory cells or smooth muscle cells). Autocrine or paracrine stimulation of cell growth caused by the effect of PDGF on cells with induced receptors may be important in the formation of the proliferative lesions found in atherosclerosis and in certain forms of chronic inflammation.

A Glioma-Derived PDGF A Chain Homodimer Has Different Functional Activities from a PDGF AB Heterodimer Purified from Human Platelets

Glioma-derived growth factor I (GDGF-I) is structurally similar to a platelet-derived growth factor (PDGF) A chain homodimer, whereas PDGF purified from human platelets is a heterodimer of one A and one B chain. Binding experiments revealed that GDGF-I and PDGF bound to a common receptor on human fibroblasts, but also suggested the presence of a second receptor type recognizing only PDGF. In contrast to PDGF, GDGF-I had only a limited mitogenic activity, a low ability to stimulate receptor autophosphorylation and actin reorganization, and no chemotactic activity. GDGF-I did, however, cause transmodulation of EGF receptors, suggesting that it, like PDGF, activates protein kinase C in fibroblasts. These data indicate that different PDGF-like growth factors have different functional activities, which are possibly mediated via different receptors.

Phosphorylation of Shc by Src family kinases is necessary for stem cell factor receptor/c-kit mediated activation of the Ras/MAP kinase pathway and c-fos induction

In this report we show that Tyr568 and Tyr570 are phosphorylated in vivo in the Kit/stem cell factor receptor (Kit/SCFR) following ligand-stimulation. By mutation of Tyr568 and Tyr570 to phenylalanine residues and expression of the mutated receptors in porcine aortic endothelial (PAE) cells, we could demonstrate a loss of activation of members of the Src family of tyrosine kinases when Tyr568 was mutated, while mutation of Tyr570 only led to a minor decrease in activation of Src family members. Mutation of both tyrosine residues led to a complete loss of Src family kinase activation. Phosphorylation of the adapter protein Shc by growth factor receptors provides association sites for Grb2-Sos, thereby activating the Ras/MAP kinase pathway. A much lowered degree of Shc phosphorylation, Ras and Erk2 activation and c-fos induction was seen in the Y568F mutant, while in the Y570F mutant these responses were less affected. In contrast, the mitogenic response was only slightly reduced. In a mutant receptor with both Tyr568 and Tyr570 mutated to phenylalanine residues, no phosphorylation of Shc and no activation of Ras and Erk2 was seen in response to stem cell factor stimulation, very weak induction of c-fos was seen and the mitogenic response was severely depressed. These data show that Ras/MAP kinase activation and c-fos induction by Kit/SCFR are mediated by members of the Src family kinases. However, the mitogenic response is only to a minor extent dependent on Src kinase activity.