Phosphorylation of Rat Insulin-like Growth Factor Binding Protein-1 Does Not Affect its Biological Properties

Peterkofsky, Beverly; Gosiewska, Anna; Wilson, Samantha M.; Kim, Yeon-Ran

doi:10.1006/abbi.1998.0797

Cited by 21 publications

(19 citation statements)

References 37 publications

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“…Rat IGFBP-1, on the other hand, is also phosphorylated, but phosphorylation does not affect IGF-binding (Peterkofsky et al 1998). Although the biologic significance of the phosphorylation may differ among species, it is a common feature of mammalian IGFBP-1.…”

Section: Discussionmentioning

confidence: 98%

Salmon serum 22 kDa insulin-like growth factor-binding protein (IGFBP) is IGFBP-1

Shimizu¹,

Dickey²,

Fukada³

et al. 2005

Journal of Endocrinology

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Western ligand blotting of salmon serum typically reveals three insulin-like growth factor (IGF) binding proteins (IGFBPs) at 22, 28 and 41 kDa. Physiologic regulation of the 22 kDa IGFBP is similar to that of mammalian IGFBP-1; it is increased in catabolic states such as fasting and stress. On the other hand, its molecular mass on Western ligand blotting is closest to mammalian IGFBP-4. The conflict between physiology and molecular mass makes it difficult to determine the identity of the 22 kDa IGFBP. This study therefore aimed to identify the 22 kDa IGFBP from protein and cDNA sequences. The 22 kDa IGFBP was purified from chinook salmon serum by a combination of IGF-affinity chromatography and reverse-phase chromatography. The N-terminal aminoacid sequence of the purified protein was used to design degenerate primers. Degenerate PCR with liver template amplified a partial IGFBP cDNA, and full-length cDNA was obtained by 5 -and 3 -rapid amplification of cDNA ends (RACE). The 1915-bp cDNA clone encodes a 23·8 kDa IGFBP, and its N-terminal amino-acid sequence matched that of purified 22 kDa IGFBP. Sequence comparison with six human IGFBPs revealed that it is most similar to IGFBP-1 (40% identity and 55% similarity). These findings indicate that salmon 22 kDa IGFBP is IGFBP-1. Salmon IGFBP-1 mRNA is predominantly expressed in the liver, and its expression levels appear to reflect circulating levels. The 3 -untranslated region of salmon IGFBP-1 mRNA contains four repeats of the nucleotide sequence ATTTA, which is involved in selective mRNA degradation. In contrast, amino-acid sequence analysis revealed that salmon IGFBP-1 does not have an Arg-Gly-Asp (RGD) integrin recognition sequence nor a Pro, Glu, Ser and Thr (PEST)-rich domain (a segment involved in rapid turnover of protein), both of which are characteristic of mammalian IGFBP-1. These findings suggest that association with the cell surface and turnover rate may differ between salmon and mammalian IGFBP-1.

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Section: Discussionmentioning

confidence: 98%

Salmon serum 22 kDa insulin-like growth factor-binding protein (IGFBP) is IGFBP-1

Shimizu¹,

Dickey²,

Fukada³

et al. 2005

Journal of Endocrinology

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“…Rat IGFBP-1 that is synthesized by H-4-II-EC3 rat hepatoma cells is phosphorylated at two sites that have been determined to be Ser 107 and Ser 132 (7). Unlike human IGFBP-1, dephosphorylation of rat IGFBP-1 does not affect its affinity for IGF-1, and dephosphorylated rat IGFBP-1 retains its capacity to inhibit DNA synthesis in 3T3 cells (7).…”

mentioning

confidence: 99%

“…In human hepatoma cells, casein kinase II and an unknown kinase that is similar but not identical to casein kinase I have been shown to phosphorylate human IGFBP-1 (6). Rat IGFBP-1 that is synthesized by H-4-II-EC3 rat hepatoma cells is phosphorylated at two sites that have been determined to be Ser 107 and Ser 132 (7). Unlike human IGFBP-1, dephosphorylation of rat IGFBP-1 does not affect its affinity for IGF-1, and dephosphorylated rat IGFBP-1 retains its capacity to inhibit DNA synthesis in 3T3 cells (7).…”

mentioning

confidence: 99%

Physiological Differences in Insulin-Like Growth Factor Binding Protein-1 (IGFBP-1) Phosphorylation in IGFBP-1 Transgenic Mice

et al. 2001

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Insulin-like growth factor binding protein (IGFBP)-1 has been shown to alter cellular responses to insulinlike growth factor 1 (IGF-1). Human IGFBP-1 undergoes serine phosphorylation, and this enhances both its affinity for IGF-1 by six-to eightfold and its capacity to inhibit IGF-1 actions. To investigate the physiological role of IGFBP-1 in vivo, transgenic mice have been generated using either the human IGFBP-1 or rat IGFBP-1 transgene. Both lines of mice expressed high concentrations of IGFBP-1 in serum and tissues; however, human IGFBP-1 transgenic mice did not show glucose intolerance and exhibited no significant intrauterine growth retardation, whereas rat IGFBP-1 transgenic mice showed fasting hyperglycemia and intrauterine growth restriction. The aim of this study was to investigate the physiological differences in the phosphorylation state of human IGFBP-1 and rat IGFBP-1 in these transgenic mice. The phosphorylation status of IGFBP-1 in transgenic mouse serum was analyzed by nondenaturing PAGE. Almost all of the IGFBP-1 in serum from the human IGFBP-1 transgenic mice was present as a nonphosphorylated form. Most of the rat IGFBP-1 in the serum of the mice expressing the rat IGFBP-1 was phosphorylated. Immunoprecipitation showed that mouse hepatoma (Hepa 1-6) cells (exposed to [ 32 P]H 3 PO 4 ) secrete 32 P-labeled IGFBP-1. When the human IGFBP-1 transgene was transfected into Hepa 1-6 cells, all of the IGFBP-1 was secreted in the nonphosphorylated form. However, when the rat IGFBP-1 transgene was transfected into these cells, phosphorylated forms of IGFBP-1 were secreted. To confirm this result, the mouse hepatoma cell protein kinase was partially purified. This kinase activity phosphorylated mouse and rat IGFBP-1 in vitro, but it did not phosphorylate human IGFBP-1. Scatchard analysis showed that the affinity of phosphorylated rat IGFBP-1 for IGF-1 was 3.9-fold higher than that of nonphosphorylated human IGFBP-1. We conclude that the mouse IGFBP-1 kinase activity cannot phosphorylate human IGFBP-1, whereas it can phosphorylate rat IGFBP-1. The phosphorylation state of human IGFBP-1 may account for part of the phenotypic differences noted in the two studies of transgenic mice, and it is an important determinant of the capacity of human IGFBP-1 to inhibit IGF-1 actions in vivo.

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“…In addition, dissection of the secretory pathway in mouse lactating mammary epithelial cells using brefeldin A has suggested the presence of two GCK activities located in either cis-or transGolgi compartments with different substrate specificities [7]. Similar results have been found for phosphorylation of insulinlike growth factor-1 binding protein-1 in 3T3 cells [20]. In other studies with brefeldin A, which have examined single substrates, opposite conclusions were reached about the site of phosphorylation in cis\medial versus trans-Golgi compartments [14,18,38].…”

Section: Discussionmentioning

confidence: 68%

“…In addition, a large number of unrelated secretory proteins with various functions have been shown to be phosphorylated during transport through the secretory pathway, including chromogranin B and secretogranin II [14], the enkephalin precursor [15], progastrin [16], osteopontin [17], sialyltransferase [18], and biologically active hormones such as prolactin [19], insulin-like growth factor-binding protein-1 [20] and many others. The significance of the phosphorylation of these proteins is generally Abbreviations used : CK casein kinase ; GCK, Golgi casein kinase.…”

Section: Introductionmentioning

confidence: 99%

Purification of Golgi casein kinase from bovine milk

Duncan

Wilkinson

Burgoyne

2000

Biochemical Journal

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Caseins and many other secretory proteins are phosphorylated during their transport through the secretory pathway by a protein kinase present within Golgi compartments. Molecular analysis of the Golgi casein kinase (GCK) has not been possible since it has not been purified to homogeneity or been cloned. Previous attempts have been made to purify GCK activity from mammary gland Golgi fractions, but these have not resulted in extensive purification of the enzyme. In the present study, we have demonstrated that substantial amounts of GCK activity, assayed using a specific peptide substrate, can be detected as a soluble form in bovine milk, and we have used milk as a source for purification. A purification protocol was established that allowed > 80000-fold purification to a specific activity of GCK (approx. 700nmoles/min per mg of protein) far higher than previously achieved. These findings cast doubts on previous claims for purification of GCK activity. In addition, ion-exchange chromotography resolved two closely eluting peaks of activity, suggesting the existence of two related, but distinct, GCK activities.

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Phosphorylation of Rat Insulin-like Growth Factor Binding Protein-1 Does Not Affect its Biological Properties

Cited by 21 publications

References 37 publications

Salmon serum 22 kDa insulin-like growth factor-binding protein (IGFBP) is IGFBP-1

Salmon serum 22 kDa insulin-like growth factor-binding protein (IGFBP) is IGFBP-1

Physiological Differences in Insulin-Like Growth Factor Binding Protein-1 (IGFBP-1) Phosphorylation in IGFBP-1 Transgenic Mice

Purification of Golgi casein kinase from bovine milk

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