Phosphorylation of PHAS-I by mitogen-activated protein (MAP) kinase. Identification of a site phosphorylated by MAP kinase in vitro and in response to insulin in rat adipocytes
PHAS-I is a heat- and acid-stable protein that is phosphorylated on Ser/Thr residues in response to insulin and growth factors. To investigate the phosphorylation of PHAS-I, the protein was expressed in bacteria and purified for use as substrate in protein kinase reactions in vitro. Recombinant PHAS...
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| Veröffentlicht in: | The Journal of biological chemistry 1994-09, Vol.269 (37), p.23185-23191 |
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| creator | T A Haystead C M Haystead C Hu T A Lin J C Lawrence, Jr |
| description | PHAS-I is a heat- and acid-stable protein that is phosphorylated on Ser/Thr residues in response to insulin and growth factors.
To investigate the phosphorylation of PHAS-I, the protein was expressed in bacteria and purified for use as substrate in protein
kinase reactions in vitro. Recombinant PHAS-I was rapidly and stoichiometrically phosphorylated by mitogen-activated protein
(MAP) kinase. At saturating MgATP, the Km and Vmax observed with PHAS-I were almost identical to those obtained with myelin
basic protein, one of the best MAP kinase substrates. PHAS-I was also phosphorylated at a significant rate by casein kinase
II and protein kinase C. To investigate sites of phosphorylation, PHAS-I was digested with collagenase and phosphopeptides
were resolved by reverse phase high performance liquid chromatography. Almost all of the phosphate introduced by MAP kinase
was recovered in the peptide, Leu-Met-Glu-Cys-Arg-Asn-Ser-Pro-Val-Ala-Lys-Thr. 32P was released in the seventh cycle of Edman
degradation, identifying the Ser (Ser64) as the phosphorylated residue. Ser64 was also phosphorylated in response to insulin
in rat adipocytes. We conclude that PHAS-I is a substrate for MAP kinase both in vivo and in vitro. As PHAS-I is one of the
most prominent insulin-stimulated phosphoproteins in adipocytes, it may qualify as the major MAP kinase substrate in these
cells. |
| doi_str_mv | 10.1016/S0021-9258(17)31637-X |
| format | Article |
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To investigate the phosphorylation of PHAS-I, the protein was expressed in bacteria and purified for use as substrate in protein
kinase reactions in vitro. Recombinant PHAS-I was rapidly and stoichiometrically phosphorylated by mitogen-activated protein
(MAP) kinase. At saturating MgATP, the Km and Vmax observed with PHAS-I were almost identical to those obtained with myelin
basic protein, one of the best MAP kinase substrates. PHAS-I was also phosphorylated at a significant rate by casein kinase
II and protein kinase C. To investigate sites of phosphorylation, PHAS-I was digested with collagenase and phosphopeptides
were resolved by reverse phase high performance liquid chromatography. Almost all of the phosphate introduced by MAP kinase
was recovered in the peptide, Leu-Met-Glu-Cys-Arg-Asn-Ser-Pro-Val-Ala-Lys-Thr. 32P was released in the seventh cycle of Edman
degradation, identifying the Ser (Ser64) as the phosphorylated residue. Ser64 was also phosphorylated in response to insulin
in rat adipocytes. We conclude that PHAS-I is a substrate for MAP kinase both in vivo and in vitro. As PHAS-I is one of the
most prominent insulin-stimulated phosphoproteins in adipocytes, it may qualify as the major MAP kinase substrate in these
cells.</description><identifier>ISSN: 0021-9258</identifier><identifier>EISSN: 1083-351X</identifier><identifier>DOI: 10.1016/S0021-9258(17)31637-X</identifier><identifier>PMID: 8083223</identifier><language>eng</language><publisher>United States: American Society for Biochemistry and Molecular Biology</publisher><subject>Adipocytes - drug effects ; Adipocytes - metabolism ; Amino Acid Sequence ; Animals ; Carrier Proteins ; Insulin - pharmacology ; Intracellular Signaling Peptides and Proteins ; Male ; Mitogen-Activated Protein Kinase 1 ; Molecular Sequence Data ; Phosphoproteins - metabolism ; Phosphorylation ; Protein-Serine-Threonine Kinases - metabolism ; Protein-Tyrosine Kinases - metabolism ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins - metabolism</subject><ispartof>The Journal of biological chemistry, 1994-09, Vol.269 (37), p.23185-23191</ispartof><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c380t-16a80d238949b01d279c13e503f67406faf4ea478723704aa39cacf8cbccab3e3</citedby><cites>FETCH-LOGICAL-c380t-16a80d238949b01d279c13e503f67406faf4ea478723704aa39cacf8cbccab3e3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>315,782,786,27933,27934</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/8083223$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>T A Haystead</creatorcontrib><creatorcontrib>C M Haystead</creatorcontrib><creatorcontrib>C Hu</creatorcontrib><creatorcontrib>T A Lin</creatorcontrib><creatorcontrib>J C Lawrence, Jr</creatorcontrib><title>Phosphorylation of PHAS-I by mitogen-activated protein (MAP) kinase. Identification of a site phosphorylated by MAP kinase in vitro and in response to insulin in rat adipocytes</title><title>The Journal of biological chemistry</title><addtitle>J Biol Chem</addtitle><description>PHAS-I is a heat- and acid-stable protein that is phosphorylated on Ser/Thr residues in response to insulin and growth factors.
To investigate the phosphorylation of PHAS-I, the protein was expressed in bacteria and purified for use as substrate in protein
kinase reactions in vitro. Recombinant PHAS-I was rapidly and stoichiometrically phosphorylated by mitogen-activated protein
(MAP) kinase. At saturating MgATP, the Km and Vmax observed with PHAS-I were almost identical to those obtained with myelin
basic protein, one of the best MAP kinase substrates. PHAS-I was also phosphorylated at a significant rate by casein kinase
II and protein kinase C. To investigate sites of phosphorylation, PHAS-I was digested with collagenase and phosphopeptides
were resolved by reverse phase high performance liquid chromatography. Almost all of the phosphate introduced by MAP kinase
was recovered in the peptide, Leu-Met-Glu-Cys-Arg-Asn-Ser-Pro-Val-Ala-Lys-Thr. 32P was released in the seventh cycle of Edman
degradation, identifying the Ser (Ser64) as the phosphorylated residue. Ser64 was also phosphorylated in response to insulin
in rat adipocytes. We conclude that PHAS-I is a substrate for MAP kinase both in vivo and in vitro. As PHAS-I is one of the
most prominent insulin-stimulated phosphoproteins in adipocytes, it may qualify as the major MAP kinase substrate in these
cells.</description><subject>Adipocytes - drug effects</subject><subject>Adipocytes - metabolism</subject><subject>Amino Acid Sequence</subject><subject>Animals</subject><subject>Carrier Proteins</subject><subject>Insulin - pharmacology</subject><subject>Intracellular Signaling Peptides and Proteins</subject><subject>Male</subject><subject>Mitogen-Activated Protein Kinase 1</subject><subject>Molecular Sequence Data</subject><subject>Phosphoproteins - metabolism</subject><subject>Phosphorylation</subject><subject>Protein-Serine-Threonine Kinases - metabolism</subject><subject>Protein-Tyrosine Kinases - metabolism</subject><subject>Rats</subject><subject>Rats, Sprague-Dawley</subject><subject>Recombinant Proteins - metabolism</subject><issn>0021-9258</issn><issn>1083-351X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1994</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpNkVFr2zAUhcXY6NJuP6GghzHaB3eSZVvyYyhbG-hYoBvkTVzL17U22_IkpSP_aj9xShPC9CLuPed8QhxCLjm74YxXnx4Zy3lW56W64vJa8ErIbPOKLDhTIhMl37wmi5PlLTkP4SdLp6j5GTlTyZTnYkH-rnsX5t753QDRuom6jq7vl4_ZijY7OtronnDKwET7DBFbOnsX0U706utyfU1_2QkC3tBVi1O0nTUnBtBgI9L5P3pKJ2TKHWM0YZ5t9I7C1O4Hj2F2UxKiS2PYDmm3X0Ok0NrZmV3E8I686WAI-P54X5AfXz5_v73PHr7drW6XD5kRisWMV6BYmwtVF3XDeJvL2nCBJRNdJQtWddAVCIVUMheSFQCiNmA6ZRpjoBEoLsjHAzf9-PcWQ9SjDQaHASZ026BlJRkvVJWM5cFovAvBY6dnb0fwO82Z3jelX5rS-xo0l_qlKb1JucvjA9tmxPaUOlaT9A8HvbdP_R_rUTfWmR5HnVe1TohccFWKf9NvnWo</recordid><startdate>19940916</startdate><enddate>19940916</enddate><creator>T A Haystead</creator><creator>C M Haystead</creator><creator>C Hu</creator><creator>T A Lin</creator><creator>J C Lawrence, Jr</creator><general>American Society for Biochemistry and Molecular Biology</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>19940916</creationdate><title>Phosphorylation of PHAS-I by mitogen-activated protein (MAP) kinase. Identification of a site phosphorylated by MAP kinase in vitro and in response to insulin in rat adipocytes</title><author>T A Haystead ; C M Haystead ; C Hu ; T A Lin ; J C Lawrence, Jr</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c380t-16a80d238949b01d279c13e503f67406faf4ea478723704aa39cacf8cbccab3e3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1994</creationdate><topic>Adipocytes - drug effects</topic><topic>Adipocytes - metabolism</topic><topic>Amino Acid Sequence</topic><topic>Animals</topic><topic>Carrier Proteins</topic><topic>Insulin - pharmacology</topic><topic>Intracellular Signaling Peptides and Proteins</topic><topic>Male</topic><topic>Mitogen-Activated Protein Kinase 1</topic><topic>Molecular Sequence Data</topic><topic>Phosphoproteins - metabolism</topic><topic>Phosphorylation</topic><topic>Protein-Serine-Threonine Kinases - metabolism</topic><topic>Protein-Tyrosine Kinases - metabolism</topic><topic>Rats</topic><topic>Rats, Sprague-Dawley</topic><topic>Recombinant Proteins - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>T A Haystead</creatorcontrib><creatorcontrib>C M Haystead</creatorcontrib><creatorcontrib>C Hu</creatorcontrib><creatorcontrib>T A Lin</creatorcontrib><creatorcontrib>J C Lawrence, Jr</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>The Journal of biological chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>T A Haystead</au><au>C M Haystead</au><au>C Hu</au><au>T A Lin</au><au>J C Lawrence, Jr</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Phosphorylation of PHAS-I by mitogen-activated protein (MAP) kinase. Identification of a site phosphorylated by MAP kinase in vitro and in response to insulin in rat adipocytes</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>1994-09-16</date><risdate>1994</risdate><volume>269</volume><issue>37</issue><spage>23185</spage><epage>23191</epage><pages>23185-23191</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><abstract>PHAS-I is a heat- and acid-stable protein that is phosphorylated on Ser/Thr residues in response to insulin and growth factors.
To investigate the phosphorylation of PHAS-I, the protein was expressed in bacteria and purified for use as substrate in protein
kinase reactions in vitro. Recombinant PHAS-I was rapidly and stoichiometrically phosphorylated by mitogen-activated protein
(MAP) kinase. At saturating MgATP, the Km and Vmax observed with PHAS-I were almost identical to those obtained with myelin
basic protein, one of the best MAP kinase substrates. PHAS-I was also phosphorylated at a significant rate by casein kinase
II and protein kinase C. To investigate sites of phosphorylation, PHAS-I was digested with collagenase and phosphopeptides
were resolved by reverse phase high performance liquid chromatography. Almost all of the phosphate introduced by MAP kinase
was recovered in the peptide, Leu-Met-Glu-Cys-Arg-Asn-Ser-Pro-Val-Ala-Lys-Thr. 32P was released in the seventh cycle of Edman
degradation, identifying the Ser (Ser64) as the phosphorylated residue. Ser64 was also phosphorylated in response to insulin
in rat adipocytes. We conclude that PHAS-I is a substrate for MAP kinase both in vivo and in vitro. As PHAS-I is one of the
most prominent insulin-stimulated phosphoproteins in adipocytes, it may qualify as the major MAP kinase substrate in these
cells.</abstract><cop>United States</cop><pub>American Society for Biochemistry and Molecular Biology</pub><pmid>8083223</pmid><doi>10.1016/S0021-9258(17)31637-X</doi><tpages>7</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | The Journal of biological chemistry, 1994-09, Vol.269 (37), p.23185-23191 |
| issn | 0021-9258 1083-351X |
| language | eng |
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| source | MEDLINE; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Alma/SFX Local Collection |
| subjects | Adipocytes - drug effects Adipocytes - metabolism Amino Acid Sequence Animals Carrier Proteins Insulin - pharmacology Intracellular Signaling Peptides and Proteins Male Mitogen-Activated Protein Kinase 1 Molecular Sequence Data Phosphoproteins - metabolism Phosphorylation Protein-Serine-Threonine Kinases - metabolism Protein-Tyrosine Kinases - metabolism Rats Rats, Sprague-Dawley Recombinant Proteins - metabolism |
| title | Phosphorylation of PHAS-I by mitogen-activated protein (MAP) kinase. Identification of a site phosphorylated by MAP kinase in vitro and in response to insulin in rat adipocytes |
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