[36] Purification and characterization of phosphorylase phosphatase from rabbit skeletal muscle

McNall, Steven J.; Ballou, Lisa M.; Villa-Moruzzi, Emma; Fischer, Edmond H.

doi:10.1016/0076-6879(88)59038-9

Cited by 5 publications

(2 citation statements)

References 23 publications

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“…Thus, 2 g cell extract was used for phosphatase activity assays. The reaction mixture contained 50 mM Tris-HCl pH 7.0, 0.1 mM EDTA, 5 mM DTT, 0.01% Brij 35®, 2 g cell extract and 4 M 32 P-MyBP (a common substrate for Ser/Thr protein phosphatases) or 7.5 M 32 P-phosphorylase-a (a specific substrate for PP1 and PP2A) in a final volume of 50 l. Reactions were carried out in a 30°C water bath for 15 minutes and processed as previously described (McNall et al, 1988). Since 1 M okadaic acid inhibits 99% of PP1 and 100% of PP2A, the combined total activitiy of PP1 and PP2A was defined as the activity sensitive to 1 M okadaic acid (Cohen, 1991).…”

Section: Protein Phosphatase Assaymentioning

confidence: 99%

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Cell adhesion regulates Ser/Thr phosphorylation and proteasomal degradation of HEF1

Zheng

McKeown‐Longo

2006

Journal of Cell Science

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Human enhancer of filamentation 1 (HEF1), a multifunctional docking protein of the Cas family, participates in integrin and growth factor signaling pathways that regulate global cellular processes including growth, motility and apoptosis. HEF1 consists of two isoforms, p105 and p115, the larger molecular weight form resulting from Ser/Thr phosphorylation of p105HEF1. The molecular mechanisms that regulate the interconversion of the two HEF1 species as well as the function of HEF1 Ser/Thr phosphorylation are unknown. Our study reveals that cell adhesion and detachment regulate the interconversion of the two HEF1 isoforms. Experiments using various inhibitors of cytoskeletal organization indicated that disruption of actin microfilaments but not intermediate filaments or microtubules resulted in a complete conversion of p115HEF1 to p105HEF1. The conversion of p115HEF1 to p105HEF1 was prevented by inhibition of protein phosphatase 2A (PP2A), suggesting that cytoskeletal regulation of PP2A activity controlled the dephosphorylation of p115HEF1. Degradation of endogenous HEF1 was dependent on proteasomes with the p115 species of HEF1 being preferentially targeted for turnover. Dephosphorylation of HEF1 by suspending cells or disrupting actin filaments protected HEF1 from degradation. These results suggest that the adhesion-dependent actin organization regulates proteasomal turnover of HEF1 through the activity of PP2A.

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Section: Protein Phosphatase Assaymentioning

confidence: 99%

“…The conversion of phosphorylase-b to phosphorylase-a was accomplished by phosphorylase kinase and [␥ -32 P]ATP, according to the procedure described by McNall et al (McNall et al, 1988).…”

Section: Preparation Of 32 P-labeled Substratesmentioning

confidence: 99%

Cell adhesion regulates Ser/Thr phosphorylation and proteasomal degradation of HEF1

Zheng

McKeown‐Longo

2006

Journal of Cell Science

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Phosphoprotein phosphatase

Schomburg¹,

Salzmann²

1991

Enzyme Handbook 3

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Phosphorylation of the catalytic subunit of type‐1 protein phosphatase by the v‐abl tyrosine kinase

Villa-Moruzzi¹,

Zonca²,

Crabb³

1991

FEBS Letters

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The catalytic subunit of type-l protein phosphatase (PPl) was phosphorylated by the tyrosine kinase v-abl as follows: (i) cytosolic PPI was phosphorylated more (0.73 molImo1) than PPI obtained from the glycogcn particles (0.076 mol/mol), while free catalytic subunit isolated in the active or inactive form from cytosolic PPI was phosphorylated even lass and catalytic subunit complexed with inhibitor-2 was not phosphorylated; (ii) phosphorylation stoichiometry was dependent on the concentration of PPI and 3 h incubation at 30°C was required for maximal phosphorylation; (iii) phosphorylation was on a tyrosine residue located in the C-terminal region of PPI which is lost during proteolysis; (iv) pb2sphorylation did not affect enzyme activity but allowed conversion from the active to the inactive form upon incubation with inhibitor-2 of a PPl form that in its dephospho-form did not convert.

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[36] Purification and characterization of phosphorylase phosphatase from rabbit skeletal muscle

Cited by 5 publications

References 23 publications

Cell adhesion regulates Ser/Thr phosphorylation and proteasomal degradation of HEF1

Cell adhesion regulates Ser/Thr phosphorylation and proteasomal degradation of HEF1

Phosphoprotein phosphatase

Phosphorylation of the catalytic subunit of type‐1 protein phosphatase by the v‐abl tyrosine kinase

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