Edwin G. Krebs

Insulin-like growth factor-I and platelet-derived growth factor-BB induce directed migration of human arterial smooth muscle cells via signaling pathways that are distinct from those of proliferation.

Directed migration or chemotaxis of arterial smooth muscle cells (SMC) contributes to intimal SMC accumulation, a key event in the development of atherosclerotic lesions and in restenosis after angioplasty. The present study compares and contrasts insulin-like growth factor I (IGF-I) and platelet-derived growth factor (PDGF-BB) as chemoattractants and mitogens for human arterial SMC. Compared with PDGF-BB, IGF-I is a weaker SMC mitogen. Thus, PDGF-BB, but not IGF-I, evokes a strong and rapid activation of mitogen-activated protein (MAP) kinase kinase and MAP kinase. However, IGF-I is a potent stimulator of directed migration of human arterial SMC, as measured in a Boyden chamber assay. The half-maximal concentration for migration is similar to the Kd for IGF-I receptor interaction. An IGF-I receptor-blocking antibody blocks the effects of IGF-I, IGF-II, and insulin, indicating that the effects are indeed mediated through the IGF-I receptor. The maximal effect of IGF-I on directed migration ranges between 50% and 100% of the effect of PDGF-BB, the strongest known chemoattractant for SMC. The ability of IGF-I and PDGF-BB to induce chemotaxis coincides with their ability to stimulate phosphatidylinositol turnover, diacylglycerol formation, and intracellular Ca2+ flux and suggests that these signaling pathways, but not activation of the MAP kinase cascade, are required for chemotaxis of human arterial SMC.

A lymphocyte-specific protein-tyrosine kinase gene is rearranged and overexpressed in the murine T cell lymphoma LSTRA

Protein-tyrosine kinases are implicated in the control of normal and neoplastic cell growth. We have used molecular cloning strategies to characterize a lymphocyte-specific protein-tyrosine kinase gene distinct from but closely related to src and yes. This gene, encoded by a genetic locus defined here as lskT, is rearranged and overexpressed in the murine T cell lymphoma LSTRA. Thus alterations in the structure or expression of this protein-tyrosine kinase gene may in some cases mediate neoplastic transformation. In addition, transcription of the normal lskT gene is restricted to cells of lymphoid origin. We infer that the lskT-encoded protein-tyrosine kinase may aid in transducing proliferative or differentiative signals unique to lymphocytes.

[43] Preparation of homogeneous cyclic AMP-dependent protein kinase(s) and its subunits from rabbit skeletal muscle

Publisher Summary Cyclic AMP (cAMP)-dependent protein kinases catalyze the transfer of the terminal phosphate of ATP to serine or threonine residues of a variety of protein substrates. These enzymes are termed cAMP-dependent becasue most if not all of their activity is revealed only in the presence of low concentrations of cAMP. The enzymes are composed of two subunit types, one of which is catalytically active (C) and another (R) which regulates the activity of the catalytic subunit. The subunit R inhibits the activity of C until cAMP is bound to R at which time the subunits dissociate and the activity of the enzyme is expressed. Currently, it appears that there is more than one cAMP dependent protein kinase present in muscle and liver. In most of the cases investigated, at least two peaks of cAMP-dependent activity can be separated by chromatography on DEAE-cellulose. These fractions will be referred to as peak I and peak II protein kinase in communication. To date, no clear-cut differences in the physiological roles of these enzyme forms have been demonstrated.

The phosphorylase b to a converting enzyme of rabbit skeletal muscle

Abstract A method for the purification of the enzyme catalyzing the conversion of phosphorylase b to a is described. After a 65-fold increase in specific activity, the enzyme obtained is free of PR enzyme activity. The course of the reaction at several concentrations of converting enzyme is illustrated, and converting enzyme units are defined. The optimum pH for the enzyme is approximately 9.0; the reaction requires Mn ++ or Mg ++ ions and ATP. It is shown that a mono-manganous-ATP complex is probably acting in the reaction. Conversion of phosphorylase b to a is carried out in the presence of 32 P-ATP, and an incorporation of at least 2 moles of 32 P per mole of phosphorylase a is found to occur.

Regulation of Oocyte Maturation

Publisher Summary Oocytes have proved to be an optimal experimental system for several lines of investigation. One such line encompasses the biochemistry of cell division, in particular the relationship of calcium and cyclic nucleotide levels to the regulation of this process. Oocytes are an attractive model for studies in this area because borders of the oocyte maturation process are physiologically arrested states of cell division, and after exposure to progesterone progress between these borders occurs in 3-5 hours without interphase. This time span and perfect physiological synchrony are useful for examining different stages in the cell division process, especially using the tool of microinjection. This ability to make additions of biochemically defined components to a single dividing cell using microinjection techniques is of special value for studies of cell division because no cell-free system exists that can carry out any of the processes involved, with the partial exception of microtubule polymerization. Another line of investigation that has increased interest in the oocyte system concerns posttranscriptional effects of steroid hormones.

p90(RSK) is a serum-stimulated Na+/H+ exchanger isoform-1 kinase. Regulatory phosphorylation of serine 703 of Na+/H+ exchanger isoform-1

The Na+/H+exchanger isoform-1 (NHE-1) is the key member of a family of exchangers that regulates intracellular pH and cell volume. Activation of NHE-1 by growth factors is rapid, correlates with increased NHE-1 phosphorylation and cell alkalinization, and plays a role in cell cycle progression. By two-dimensional tryptic peptide mapping of immunoprecipitated NHE-1, we identify serine 703 as the major serum-stimulated amino acid. Mutation of serine 703 to alanine had no effect on acid-stimulated Na+/H+ exchange but completely prevented the growth factor-mediated increase in NHE-1 affinity for H+. In addition, we show that p90 ribosomal S6 kinase (p90RSK) is a key NHE-1 kinase since p90RSK phosphorylates NHE-1 serine 703 stoichiometricallyin vitro, and transfection with kinase-inactive p90RSK inhibits serum-induced phosphorylation of NHE-1 serine 703 in transfected 293 cells. These findings establish p90RSK as a serum-stimulated NHE-1 kinase and a mediator of increased Na+/H+ exchange in vivo.

[49a] Muscle phosphorylase b: x Glucose-1-phosphate+ Gn ⇄ Gn+x + x inorganic phosphate(where Gn designates glycogen containing n glucose residues)

Publisher Summary This chapter describes an assay method, purification procedure, and properties of muscle phosphorylase b . The assay is carried out as described in the presence of adenosine monophosphate (AMP), since phosphorylase b is enzymatically inactive in the absence of this nucleotide. Phosphorylase b is crystallized by the addition of AMP and magnesium acetate to a partially purified ammonium sulfate fraction of muscle extract. The extraction procedure is designed to give large yields of phosphorylase and to make certain that all this enzyme is in the form of phosphorylase b . These extracts are not suitable for conversion of the enzyme to the a form at the crude extract stage, but the isolated phosphorylase b can be readily converted to phosphorylase a with purified phosphorylase b kinase. Phosphorylase b is enzymatically inactive in the absence of AMP. Crystalline phosphorylase b is homogeneous in the electrophoresis apparatus and the ultracentrifuge. Phospborylase b is much more soluble than phosphorylase a in a glycerophosphate-cysteine buffer, pH 7, and does not crystallize spontaneously like this latter enzyme, even at a concentration of 50 mg/ml at 0°.

The mitogen-activated protein kinase activator

The mitogen-activated protein kinase appears to be regulated by another growth factor regulated kinase, the mitogen-activated protein kinase activator. In the past year, much progress has been made in purifying and characterizing the mitogen-activated protein kinase activator, in determining its primary structure, and in identifying another protein kinase that may function upstream to regulate its activity.

Neoplastic transformation induced by an activated lymphocyte-specific protein tyrosine kinase (pp56lck)

The lck proto-oncogene encodes a lymphocyte-specific member of the src family of protein tyrosine kinases. Here we demonstrate that pp56lck is phosphorylated in vivo at a carboxy-terminal tyrosine residue (Tyr-505) analogous to Tyr-527 of pp60c-src. Substitution of phenylalanine for tyrosine at this position resulted in increased phosphorylation of a second tyrosine residue (Tyr-394) and was associated with an increase in apparent kinase activity. In addition, this single point mutation unmasked the oncogenic potential of pp56lck in NIH 3T3 cell transformation assays. Viewed in the context of similar results obtained with pp60c-src, it is likely that the enzymatic activity and transforming ability of all src-family protein tyrosine kinases can be regulated by carboxy-terminal tyrosine phosphorylation. We further demonstrate that overexpression of pp56lck in the murine T-cell lymphoma LSTRA as a result of a retroviral insertion event produces a kinase protein that despite wild-type primary structure is nevertheless hypophosphorylated at Tyr-505. Thus, control of normal growth in this lymphoid cell line may have been abrogated through acquisition of a posttranslationally activated version of pp56lck.

The mitogen-activated protein kinase pathway can mediate growth inhibition and proliferation in smooth muscle cells. Dependence on the availability of downstream targets.

Activation of the classical mitogen-activated protein kinase (MAPK) pathway leads to proliferation of many cell types. Accordingly, an inhibitor of MAPK kinase, PD 098059, inhibits PDGF-induced proliferation of human arterial smooth muscle cells (SMCs) that do not secrete growth-inhibitory PGs such as PGE2. In striking contrast, in SMCs that express the inducible form of cyclooxygenase (COX-2), activation of MAPK serves as a negative regulator of proliferation. In these cells, PDGF-induced MAPK activation leads to cytosolic phospholipase A2 activation, PGE2 release, and subsequent activation of the cAMP-dependent protein kinase (PKA), which acts as a strong inhibitor of SMC proliferation. Inhibition of either MAPK kinase signaling or of COX-2 in these cells releases them from the influence of the growth-inhibitory PGs and results in the subsequent cell cycle traverse and proliferation. Thus, the MAPK pathway mediates either proliferation or growth inhibition in human arterial SMCs depending on the availability of specific downstream enzyme targets.