Phosphorylation of a Novel Myosin Binding Subunit of Protein Phosphatase 1 Reveals a Conserved Mechanism in the Regulation of Actin Cytoskeleton

Abstract

The myotonic dystrophy kinase-related kinases RhoA binding kinase and myotonic dystrophy kinase-related Cdc42 binding kinase (MRCK) are effectors of RhoA and Cdc42, respectively, for actin reorganization. Using substrate screening in various tissues, we uncovered two major substrates, p130 and p85, for MRCK␣-kinase. p130 is identified as myosin binding subunit p130, whereas p85 is a novel related protein. p85 contains N-terminal ankyrin repeats, an ␣-helical C terminus with leucine repeats, and a centrally located conserved motif with the MRCK␣-kinase phosphorylation site. Like MBS130, p85 is specifically associated with protein phosphatase 1␦ (PP1␦), and this requires the N terminus, including the ankyrin repeats. This association is required for the regulation of both the catalytic activities and the assembly of actin cytoskeleton. The N terminus, in association with PP1␦, is essential for actin depolymerization, whereas the C terminus antagonizes this action. The C-terminal effects consist of two independent events that involved both the conserved phosphorylation inhibitory motif and the ␣-helical leucine repeats. The former was able to interact with PP1␦ only in the phosphorylated state and result in inactivation of PP1␦ activity. This provides further evidence that phosphorylation of a myosin binding subunit protein by specific kinases confers conformational changes in a highly conserved region that plays an essential role in the regulation of its catalytic subunit activities.

PDZ Domain-mediated Interaction of Interleukin-16 Precursor Proteins with Myosin Phosphatase Targeting Subunits

“…In this context a functional link to the chemotactic activity of the cytokine would exist. Alternatively, as has been shown with MBS85, treatment with lysophosphatidic acid and phorbol esters leads to a rapid translocation of the protein to the peripheral membrane (19), suggesting an involvement in activation-induced cortical rearrangements or targeting of cytoplasmic or recycling proteins to the membrane. Several lines of evidence indicate that cellular motor proteins, including myosins, are key players in the trafficking of receptors and ion channels to the cellular membrane as well as in the opposite direction during endocytosis and protein sorting events (44 -46).…”

“…The pCMV/ MYPT2-(657-982), pCMV/MYPT2-(657-978), and pYesTrp2/MYPT1-(904 -1030) constructs were made by PCR amplification of the indicated subsequence of MYPT2 or MYPT1 and insertion into pCMV3-Tag3B and pYesTrp2, respectively. The pXJ-FLAG-MBS85 plasmid for the expression of FLAG-MBS85 was contributed by Thomas Leung (Glaxo-IMCB Group, Singapore) and has been described previously (19).…”

Phosphoprotein inhibitor CPI-17 specificity depends on allosteric regulation of protein phosphatase-1 by regulatory subunits

“…However, this does not imply that MYPT1-containing phosphatase is the only CPI-17-sensitive holoenzyme. Related regulatory subunits such as p85 (33), or other subunits, may also produce the conformation of PP1C that binds phospho-CPI-17 with high affinity and restricted catalytic activity. These would be CPI-17-sensitive by the same proposed mechanism.…”

Identification of a Tankyrase-binding Motif Shared by IRAP, TAB182, and Human TRF1 but Not Mouse TRF1

“…These clones encoded fragments of seven proteins (overlapping fragments in three cases), each fused in-frame to the GAL4 activation domain. Interestingly, the RXXPDG motif was found in four of these proteins: NuMA, Hox-B2 (a homeobox protein) (15), a regulatory subunit of the protein phosphatase PP1␦ (16), and the L-type calcium channel (in the cytosolic tail of its ␣1 subunit) (17). Two additional proteins matched the motif except at position 4 (as in hTRF1): the deubiquitinating enzyme USP25 (18) and the Tax1-binding protein TXBP151 (19).…”