"DE RecName: Full=Serine/threonine-protein kinase LMTK2;"

"CC -!- FUNCTION: Phosphorylates PPP1C, phosphorylase b and CFTR."

"CC Q8IWU2; P36873: PPP1CC; NbExp=9; IntAct=EBI-2008933, EBI-356283;"

"CC Q8IWU2; P41236: PPP1R2; NbExp=4; IntAct=EBI-2008933, EBI-1056517;"

"KPI-2 is a 1503-residue protein with two predicted transmembrane helices at the N terminus, a kinase domain, followed by a C-terminal domain. The transmembrane helices were sufficient for targeting proteins to the membrane."

"However, it only exhibited serine/threonine kinase activity in autophosphorylation reactions or with added substrates."

"KPI-2 kinase domain phosphorylated protein phosphatase-1 (PP1C) at Thr(320), which attenuated PP1C activity. KPI-2 C-terminal domain directly associated with PP1C, and this required a VTF motif. Inh2 associated with KPI-2 C-terminal domain with and without PP1C."

"Cprk interacts with p35 in the yeast-two hybrid system, binds to p35 in glutathione S-transferase fusion pull-down assays, and colocalizes with p35 in cultured neurons and transfected cells. In these cells, cprk is present with p35 in the Golgi apparatus."

"Cprk displays catalytic activity in in vitro kinase assays and is itself phosphorylated by cdk5/p35. Cdk5/p35 inhibits cprk activity."

"We found that KPI-2 is strictly a Ser/Thr kinase that reacts with Ser either preceded by or followed by Pro residues but unlike other Pro-directed kinases does not strictly require an adjacent Pro residue."

"The most reactive peptide in the library corresponds to Ser-737 of cystic fibrosis transmembrane conductance regulator, and the recombinant R domain of cystic fibrosis transmembrane conductance regulator was a preferred substrate."

"Phosphorylase was used as a specific substrate to show that KPI-2 is inhibited in living cells by addition of nerve growth factor or serum."

"In this paper we have identified a new myosin-VI-binding partner, lemur tyrosine kinase 2 (LMTK2), which is the first transmembrane protein and kinase that directly binds to myosin VI. LMTK2 binds to the WWY site in the C-terminal myosin VI tail, the same site as the endocytic adaptor protein Dab2."

"When either myosin VI or LMTK2 is depleted by siRNAs, the transferrin receptor (TfR) is trapped in swollen endosomes and tubule formation in the endocytic recycling pathway is dramatically reduced, showing that both proteins are required for the transport of cargo, such as the TfR, from early endosomes to the endocytic recycling compartment."

"Double labelling experiments show that LMTK2 is present on a subset of early endosomes as revealed by colocalisation with Rab5 and EEA1 (Fig. 4, d-j) and also associated with the Rab11-positive endocytic recycling compartment (Fig. 4, k-m)."

"Several binding experiments confirmed the interaction of myosin VI with BREK in vivo and in vitro."

"Immunocytochemical analyses revealed that BREK localizes to cytoplasmic membrane vesicles and to perinuclear recycling endosomes."

"Notably, cells in which BREK was depleted by siRNA were still able to internalize transferrin molecules and to transport them to early endosomes, but were unable to transport them to perinuclear recycling endosomes."

"The resulting high-resolution network model of 480 protein-protein interactions among 270 network components suggests participation of Hippo pathway components in three distinct modules that all converge on the transcriptional co-activator YAP1."

"Using quantitative proteomics, we detect specific interactions, estimate interaction stoichiometries, and measure cellular abundances of interacting proteins."

"However, it should be noted that these interactions are not equivalent to enzyme-substrate interactions, and therefore do not necessarily suggest that the involved phosphatase directly dephosphorylates a given RTK or, conversely, that the RTK can phosphorylate the phosphatase."

"Here, we used quantitative affinity proteomics to assay protein-protein interactions for 54 phosphatases distributed across the three major protein phosphatase families, with additional analysis of their 12 co-factors."

"Through affinity-purification mass spectrometry, we have created two proteome-scale, cell-line-specific interaction networks."

"We use affinity purification coupled to mass spectrometry (AP-MS) to characterize stable binding partners and RTK-protein complexes, proximity-dependent biotin identification (BioID) to identify transient and proximal interactions, and an in vitro kinase assay to identify RTK substrates."

"Here we construct a global map of human subcellular architecture through joint measurement of biophysical interactions and immunofluorescence images for over 5,100 proteins in U2OS osteosarcoma cells."

"Our results indicate that LMTK2 is an integral membrane protein in which both the amino and carboxyl termini are exposed to the cytoplasm. Moreover, this topology places the kinase active site within the cytoplasm."

"In conclusion, we have, for the first time, formally demonstrated the topology and orientation of LMTK2 within endosomal membranes."

"Here we demonstrate that LMTK2 co-immunoprecipitates with CFTR and phosphorylates CFTR-Ser(737) in human airway epithelial cells."

"LMTK2 knockdown or expression of inactive LMTK2 kinase domain increases cell surface density of CFTR by attenuating its endocytosis in human airway epithelial cells."

"Mutation of the di-acidic export motif led to ER retention of LMTK2, and an increase in protein half-life and a concomitant loss of LMTK2 from its appropriate terminal destination. Loss of LMTK2 from endosomal compartments by preventing its release from the ER is linked to a reduction in transferrin recycling."

"Here, we demonstrate that LMTK2 is phosphorylated on serine-1418 (LMTK2ser¹⁴¹⁸) by cdk5/p35 and present evidence that this regulates its ability to phosphorylate PP1Cthr³²⁰."

"Binding to KLC1/2 involves a C-terminal tryptophan/aspartate (WD) motif in LMTK2 and the tetratricopeptide repeat (TPR) domains in KLC1/2, and this interaction facilitates axonal transport of LMTK2."

"We also show that LMTK2 facilitates the formation of a complex containing KLC1 and p35 and that siRNA loss of LMTK2 disrupts axonal transport of both p35 and cdk5."

"Detailed histological analysis revealed that Brek(-/-) germ cells differentiated normally until the round-spermatid stage, but failed to undergo the normal change in morphology to become elongated spermatids."

"On the basis of these findings, we concluded that Brek is essential for a late stage of spermatogenesis."

"LMTK2 signals via protein phosphatase-1C (PP1C) to increase inhibitory phosphorylation of GSK3β on serine-9 that reduces KLC2 phosphorylation and promotes binding of the known KLC2 cargo Smad2."

"We show that small interfering RNA loss of LMTK2 not only reduces binding of Smad2 to KLC2, but also inhibits TGFβ-induced Smad2 signalling."

"Second, TGF-β1 inhibited PP1c by an LMTK2-dependent mechanism. Third, TGF-β1 used LMTK2 to activate canonical Smad3-mediated signaling."

"However, experimental data conducted by several laboratories independently have proved that LMTK1, LMTK2 and LMTK3 solely phosphorylate serine and threonine residues and do not target tyrosine residue"

"For example, LMTK2 is a membrane-anchored serine/threonine kinase formerly known as kinase/phosphatase/inhibitor-2 (KPI-2), Cdk5/p35-regulated kinase (Cprk), brain-enriched kinase (BREK), apoptosis-associated tyrosine kinase-2 (AATYK2), KIAA1079 and LMR2"