Protein Kinase D1 Autophosphorylation via Distinct Mechanisms at Ser744/Ser748 and Ser916*

Protein kinase D1 (PKD1) is a physiologically important signaling enzyme that is activated via protein kinase C-dependent trans-phosphorylation of the activation loop at Ser744 and Ser748 followed by PKD1 autophosphorylation at Ser916. Although PKD-Ser916 autophosphorylation is widely used to track cellular PKD activity, this study exposes conditions leading to increased PKD-Ser(P)916 immunoreactivity without an associated increase in PKD activity in cardiomyocytes that heterologously overexpress catalytically inactive PKD1 and in cardiomyocytes treated with Gö6976 (a PKD inhibitor that competes with ATP). In each case, PKD1 is detected as a Ser916-phosphorylated enzyme that lacks kinase activity. In vitro kinase assays reconcile these seemingly discrepant findings by demonstrating that PKD1-Ser916 autophosphorylation can proceed via either an intermolecular reaction or an intramolecular autophosphorylation that requires only very low ATP concentrations that do not support target substrate phosphorylation. Additional studies show that Ser744 and Ser748 are targets for a protein kinase C-independent autocatalytic phosphorylation and that the PKD1-S744A/S748A mutant is a Ser916-phosphorylated enzyme that is not active toward heterologous substrates. In contrast, PKD1-S916A is an active kinase that autophosphorylates at Ser744. However, the S916A substitution leads to a Ser748 phosphorylation defect and a prolonged cellular PKD1 signaling response. Collectively, these results implicate PKD1-Ser744 phosphorylation in the phorbol 12-myristate 13-acetate-dependent mechanism that increases PKD1 activity toward physiologically relevant substrates. We show that PKD1-Ser916 autophosphorylation does not necessarily correlate with PKD1 activity. Rather, autophosphorylation at Ser916 is required for subsequent autophosphorylation at Ser748. Finally, this study exposes a novel role for Ser916 and/or Ser748 autophosphorylation to terminate the cellular PKD1 signaling response.

Protein kinase D1 (PKD1) 2 is the founding member of a family of three related serine/threonine kinases that share a similar structural architecture and control a large number of biological pro-cesses that influence cell growth, differentiation, migration, and apoptosis (1,2). PKDs have an N-terminal regulatory domain containing tandem cysteine-rich C1A/C1B domains that bind diacylglycerol-/phorbol ester-enriched membranes with high affinity and a pleckstrin homology (PH) domain that participates in protein-protein interactions. PH domain-dependent autoinhibitory intramolecular interactions maintain the enzyme in an inactive state, with low basal activity, in resting cells. PKD isoforms are activated by agonists that promote diacylglycerol accumulation and activate novel PKC (nPKC) isoforms at membranes. nPKCs activate PKD isoforms by phosphorylating a pair of highly conserved serine residues (Ser 744 /Ser 748 in PKD1, nomenclature based upon rodent sequence) in the kinase domain activation loop. This post-translational modification relieves autoinhibition and stabilizes the activation loop in a conformation that is optimized for catalysis. PKD1 then undergoes a series of autophosphorylation reactions at a cluster of serine residues at Ser 205 /Ser 208 and Ser 219 /Ser 223 in the regulatory C1A/C1B interdomain region and at Ser 916 at the extreme C terminus. These autophosphorylation reactions create docking sites for PKD1 binding partners and influence PKD1 localization within the cell (3,4). A recent study also identified PKD1 autophosphorylation at the activation loop (primarily at Ser 748 ) during the chronic phase of PKD1 activation in bombesin-treated COS-7 cells (5); the relative roles of autocatalytic versus PKC-dependent activation loop phosphorylation in other cellular contexts has never been considered.
functionally distinct phosphorylation clusters at Ser 23 /Ser 24 , Ser 43 /Ser 45 , and Thr 144 . PKD-dependent cTnI phosphorylation has been mapped to Ser 23 /Ser 24 , a modification that desensitizes the myofilament to Ca 2ϩ and leads to functionally important changes in contractile performance (2,11,12). Finally, PKD phosphorylates heat shock protein 27 (HSP27); a PKD-HSP27 phosphorylation pathway has been implicated in the vascular endothelial growth factor-dependent angiogenic response (13,14). PKD1 phosphorylation at Ser 916 is viewed as an obligatory autocatalytic reaction. Immunoblotting with a phosphorylation site-specific antibody (PSSA) that recognizes PKD-Ser 916 phosphorylation is widely used as a convenient surrogate method to track PKD activity (in place of more cumbersome direct enzyme activity measurements). This approach is based upon early studies showing that phorbol ester-dependent PKD activation is accompanied by an increase in PKD-Ser 916 phosphorylation, that constitutively active forms of PKD1 (such as the PH domain-deleted or S744E/S748E-substituted mutants) are constitutively Ser 916 -phosphorylated under resting conditions in several cell types, and that catalytically inactive PKD1-D733A and K618M mutants display a Ser 916 phosphorylation defect in some experimental models (15). The notion that PKD-Ser 916 phosphorylation faithfully reports PKD activity in cells has generally remained unchallenged, although the literature is littered with isolated reports hinting that this conclusion may not necessarily be valid under all experimental conditions (16 -18). This study exposes differences in the control of PKD1-Ser 916 phosphorylation and PKD1 activity in cardiomyocytes, further undermining the assumption that PKD-Ser 916 immunoreactivity faithfully reports PKD activation in all cellular contexts. We also identify a PKD1 activation loop Ser 744 and Ser 748 autophosphorylation mechanism that might assume functional importance when nPKC isoforms are down-regulated. Finally, we identify a novel role for PKD1-Ser 916 autophosphorylation to prime PKD1 for a subsequent autophosphorylation reaction at Ser 748 . Although autophosphorylation reactions at Ser 748 and/or Ser 916 are not required for PMA-dependent PKD1 activation, this study exposes a novel role for Ser 916 and/or Ser 748 autophosphorylation to limit the duration of the cellular PKD1 signaling response.
Cardiomyocyte Culture-Cardiomyocytes were isolated from hearts of 2-day-old Wistar rats by a trypsin dispersion pro-cedure that uses a differential attachment procedure followed by irradiation to enrich for cardiomyocytes (19). Cells were plated on protamine sulfate-coated culture dishes at a density of 5 ϫ 10 6 cells/100-mm dish and grown in minimum Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum for 4 days and then serum-deprived for 24 h prior to experiments.
Adenoviral Infections-Cardiomyocytes were infected with adenoviral constructs that drive expression of HA-tagged kinase-dead PKD1 (KD-PKD1, generously provided by Drs. Terry Rogers and William Randall, University of Maryland) or ␤-galactosidase as a control. Infections were performed on cultures grown for 5 days in minimum Eagle's medium supplemented with 10% fetal calf serum (with protein extracts prepared 48 h following infections) according to methods described previously (6).
PKD1 Mutants-Plasmids that drive expression of HAtagged WT-PKD1, PKD1-SS/EE (harboring phosphomimetic substitutions at Ser 744 /Ser 748 in the activation loop), PKD1-SS/AA (harboring nonphosphorylatable alanines at Ser 744 / Ser 748 in the activation loop), PKD1-⌬PH (PH domain deletion), PKD1-⌬C1 (an N-terminal deletion mutant that lacks the C1 domain), and PKD1-S916A generated by the Toker laboratory were obtained from Addgene. A plasmid that drives expression of GFP-tagged full-length PKD1 was generously provided by Drs. David E. Clapham and Elena Oancea and has been described previously (20). PKD1 expression plasmids were introduced into COS-7 or HEK293 cells by Effectene transfection reagent (Qiagen) according to the instruction manual. Cells were grown for 24 h and lysed in RIPA buffer containing 1 mM sodium orthovanadate, 10 g/ml aprotinin, 10 g/ml leupeptin, 10 g/ml benzamidine, 0.5 mM phenylmethylsulfonyl fluoride, 5 M pepstatin A, and 0.1 M calyculin. Cell extracts were subjected to immunoprecipitation with anti-HA tag-agarose (Roche Applied Science).
In Vitro Kinase Assays-In vitro kinase assays were performed with 0.1 g of recombinant human PKD1 or with PKD1 immunoprecipitated from 150 g of starting cell extract. Incubations were performed in 110 l of a reaction buffer containing 30 mM Tris-Cl, pH 7.5, 5.45 mM MgCl 2 , 0.65 mM EDTA, 0.65 mM EGTA, 0.1 mM dithiothreitol, 1.09 mM sodium orthovanadate, 0.1 M calyculin, 0.55 M protein kinase inhibitor, 217 mM NaCl, 3.6% glycerol, and [␥-32 P]ATP (10 Ci, 66 M, unless indicated otherwise). The reaction buffer used for in vitro kinase assays depicted in Figs. 3-7 and Fig. 9 contained 89 g/ml phosphatidylserine (PS) and 175 nM PMA. Assay buffer used in Fig. 8 contained 89 g/ml PS plus 175 nM PMA, 89 g/ml PS plus 200 nM PDBu, or 30 g/ml dextran sulfate as indicated. Assays contained either 4 g of troponin complex (consisting of equimolar concentrations of cardiac troponin I, cardiac troponin T, and cardiac troponin C, generously provided by Drs. John Solaro and Marius Sumandea) or recombinant human CREB-maltose binding protein fusion construct (CREB-MBP, 1 g per assay, BIOSOURCE). Incubations were for 30 min at 30°C. PKD1 autophosphorylation was tracked with the Cell Signaling Technology anti-PKD1-Ser(P) 744 / Ser(P) 748 (which is reported to track primarily Ser 744 phosphorylation), the Cell Signaling Technology anti-PKD1-Ser(P) 916 PSSA, and the Abcam anti-PKD1-Ser(P) 742 antibody (which was recently characterized as relatively selective for Ser 742 in human PKD1, corresponding to Ser 748 in rodent PKD1 (5)).
Syntide-2 kinase assays were performed in a similar manner, with 0.36 mg/ml syntide-2 included as the PKD1 substrate. Assays were terminated by adding 30 l of 350 mM phosphoric acid followed by centrifugation at 14,000 ϫ g for 10 min. 50 l of each supernatant was spotted onto phosphocellulose filter papers (P-81), dropped into 75 mM phosphoric acid, washed (three times for 5 min), dried, and counted for radioactivity.

RESULTS
KD-PKD1 Is Phosphorylated in Trans at Ser 916 -Cardiomyocytes that heterologously overexpress KD-PKD1 at levels ϳ8-fold higher than endogenous PKD1 were treated with PMA (a direct activator of PKD and phorbol ester-sensitive PKC isoforms) as an initial strategy to resolve PKD1 phosphorylation via an obligate intramolecular autophosphorylation reaction from PKD1 phosphorylations in trans by some other cellular Ser/Thr kinase. PKD1 phosphorylation was tracked by immunoblot analysis with a PSSA that recognizes phosphorylation at Ser 916 and two PSSAs that recognize phosphorylation at the activation loop. We used a PSSA from Cell Signaling Technology (Cell Signaling Technology) that is designated anti-PKD-Ser(P) 744 /Ser(P) 748 . This PSSA was raised against a peptide phosphorylated on serines equivalent to Ser 744 and Ser 748 in rodent PKD1. However, the Rozengurt laboratory has reported that this PSSA primarily recognizes PKD1-Ser 744 phosphorylation. We also used an Abcam anti-PKD1-Ser(P) 748 PSSA (nomenclature based upon rodent sequence) that preferentially recognizes PKD1 phosphorylation at Ser 748 (5). Preliminary experiments validated the specificity of this reagent, showing that the Abcam anti-PKD1-Ser(P) 748 PSSA specifically recognizes PMA-dependent phosphorylation of WT-PKD1, but it does not recognize the PMA-activated PKD1-S744A/S748A or PKD1-S744E/S748E enzymes (data not shown). Conditions were optimized so that phosphorylation of both endogenous PKD and the KD-PKD1 transgene could be detected in a single experiment by varying gel exposure. Fig. 1 shows that PMA treatment leads to a robust increase in endogenous PKD phosphorylation that is recognized by the Cell Signaling Technology anti-PKD1-Ser(P) 744 /Ser(P) 748 , the Abcam anti-PKD1-Ser(P) 748 , and the anti-PKD1-Ser(P) 916 PSSAs (lanes 1 and 2). The PMA-activated PKD1 enzyme also migrates more slowly in SDS-PAGE. Heterologously overexpressed KD-PKD1 also is recognized by the anti-PKD-Ser(P) 744 /Ser(P) 748 and anti-PKD1-Ser(P) 916 PSSAs in resting cardiomyocytes (Fig. 1, lane 5). This cannot be dismissed as an effect of these PSSAs to recognize KD-PKD1 in a phosphorylation-independent manner at high levels of transgene overexpression, because anti-PKD1-Ser(P) 744 /Ser(P) 748 and anti-PKD1-Ser(P) 916 immunoreactivity increases further (to a level that exceeds immunoreactivity in basal Ad-KD-PKD1 cultures or PMA-treated Ad-␤-gal cultures) when Ad-KD-PKD1 cultures are treated with PMA (Fig. 1, lane 6). Because KD-PKD1 cannot execute a cis autophosphorylation reaction, KD-PKD1 must be phosphorylated at Ser 744 /Ser 748 and Ser 916 in trans by an endogenous cellular Ser/Thr kinase.
A comparison of KD-PKD1 phosphorylation at individual activation loop sites also is revealing. Fig. 1 shows that the Cell Signaling Technology anti-PKD1-Ser(P) 744 /Ser(P) 748 PSSA detects a low level of KD-PKD1 phosphorylation in resting cardiomyocytes; KD-PKD1-Ser 744 /Ser 748 phosphorylation is markedly increased by PMA. In contrast, the anti-PKD1-Ser(P) 748 PSSA detects KD-PKD1 in PMA-treated, but not resting, cardiomyocytes. Although anti-PKD1-Ser(P) 748 immunoreactivity is higher in PMA-treated Ad-KD-PKD1 cultures than in PMA-treated Ad-␤-gal cultures, the increment in anti-PKD1-Ser(P) 748 immunoreactivity because of KD-PKD1 overexpression is quite modest, given the high level of transgene overexpression (Fig. 1, compare  lanes 6 and 4). These results provided the first hint that PKD1 phosphorylation at Ser 744 and Ser 748 might be controlled via distinct molecular mechanisms (and that an autophosphorylation mechanisms is required for optimal PKD1 phosphorylation at Ser 748 but not Ser 744 ).
Gö6976 Does Not Block PKD-Ser 916 Phosphorylation in Vivo in Cardiomyocytes-PKD-Ser 916 phosphorylation mechanisms also were interrogated with pharmacologic inhibitors. We recently reported that PKD is a PKC␦-activated enzyme that functions downstream from ␣ 1 -adrenergic receptors as a CREB-Ser 133 kinase in cardiomyocytes (6). Fig. 2A replicates some of the findings from the previous study, showing that the ␣ 1 -adrenergic receptor agonist norepinephrine (NE) and PMA promote PKD phosphorylation at Ser 744 /Ser 748 and Ser 916 , and this is associated with an increase in CREB phosphorylation at Ser 133 . NE-and PMA-dependent CREB-Ser 133 phosphoryla- tion is inhibited by GF109203X, a PKC inhibitor that prevents PKC-dependent PKD activation (but does not directly inhibit PKD activity). NE-and PMA-dependent CREB-Ser 133 phosphorylation also is inhibited by Gö6976, a PKD inhibitor that does not inhibit nPKC isoforms. In contrast, whereas previous studies demonstrated that NE and PMA activate p90 ribosomal S6 kinase via a MEK-ERK-dependent pathway that is abrogated by the MEK inhibitor U0126, and p90 ribosomal S6 kinase has been implicated as a CREB-Ser 133 kinase in many cell types, NE-and PMA-dependent CREB-Ser 133 phosphorylation is not blocked by U0126 in cardiomyocytes ( Fig. 2A) (6). Fig. 2 shows that the PKC inhibitor GF109203X prevents agonist-dependent PKD phosphorylation at Ser 744 /Ser 748 and Ser 916 , consistent with the notion that PKD activation is initiated by PKC-dependent PKD-Ser 744 /Ser 748 phosphorylation mechanism that increases PKD activity and leads to subsequent PKD autophosphorylation at Ser 916 . Gö6976 does not block NE-or PMA-dependent PKD-Ser 744 /Ser 748 phosphorylation, as predicted by prevailing models that attribute PKD-Ser 744 / Ser 748 phosphorylation to an nPKC isoform; nPKC isoforms are not inhibited by Gö6976 (6). However, Fig. 2 provides surprising evidence that a biologically relevant concentration of Gö6976, which inhibits PKD-dependent trans-phosphorylation of CREB at Ser 133 , does not inhibit PKD-Ser 916 phosphorylation. This result was unanticipated; we had planned to use immunoblotting for PKD-Ser 916 phosphorylation as a convenient method to assess the efficacy of PKD inhibition by Gö6976. The divergent controls for PKD-Ser 916 and CREB-Ser 133 phosphorylation identified in our study argue that PKD-Ser 916 phosphorylation does not necessarily provide a valid read-out of PKD activity in Gö6976-treated cardiomyocytes.
In Vitro Kinase Assays Identify Distinct ATP Requirements for PKD1 Autophosphorylation at Ser 916 and PKD1 Phosphorylation of Heterologous Substrates-Because Gö6976 acts as a competitive inhibitor of ATP, we reasoned that a PKD1-Ser 916 auto-phosphorylation reaction might be relatively resistant to inhibition by Gö6976 if its ATP requirement was low, relative to the ATP requirement for PKD-dependent phosphorylation of CREB. We compared the ATP requirements for PKD1 autophosphorylation at Ser 916 and PKD1 phosphorylation of target substrates in trans to test this hypothesis. In vitro kinase assays were performed in parallel with either CREB or cTnI as substrate to examine PKD1 activity toward two physiologically relevant substrates. Immunoblot analysis was performed with PSSAs that track PKD1-Ser 916 autophosphorylation, PKD1-dependent CREB-Ser 133 phosphorylation, and PKD1-dependent cTnI-Ser 23 /Ser 24 phosphorylation. Given recent evidence that a autocatalytic reaction may contribute to PKD1 activation loop autophosphorylation (5), PKD1 autophosphorylation also was examined using the Cell Signaling Technology anti-PKD-Ser(P) 744 /Ser(P) 748 PSSA that preferentially recognizes phosphorylation at Ser 744 and the Abcam anti-PKD-Ser(P) 748 PSSA. Fig. 3A shows that PKD1 executes autophosphorylation reactions at the activation loop (at both Ser 744 and Ser 748 ) and at Ser 916 with markedly different ATP requirements. PKD-Ser 916 autophosphorylation is detected at 1 M ATP; the threshold concentration for PKD-Ser 916 autophosphorylation is ϳ0.3 M (data not shown). PKD-Ser 916 autophosphorylation does not increase at higher ATP concentrations, although the anti-PKD-Ser(P) 916 PSSA detects a pronounced phosphorylation-dependent mobility shift as the ATP concentration increases to 6 -66 M. PKD autophosphorylations at Ser 744 or Ser 748 are not detected at 1 M ATP; PKD1-Ser 744 and Ser 748 autophosphorylations are detected at 6 M ATP and maximal at 66 M ATP. These results suggest that the ATP requirement for PKD autophosphorylation at Ser 916 is considerably lower than the ATP requirement for PKD autophosphorylation at Ser 744 or Ser 748 . However, the apparent differences in PKD1 autophosphorylation at Ser 916 versus Ser 744 /Ser 748 in Fig. 3A could be a feature of the immunoblotting method, if the PSSAs used in our study recognize individual phosphorylation reactions with markedly different affinities. Therefore, the in vitro kinase assays were performed with buffers containing [ 32 P]ATP, and 32 P incorporation into PKD1 was quantified by PhosphorImager (a highly sensitive detection method that is not undermined by the uncertainties inherent in immunoblotting with a panel of PSSAs). Fig. 3B shows that overall PKD1 autophosphorylation (detected as 32 P incorporation into PKD1) increases in a dose-dependent manner as the ATP concentration is increased from 6 to 200 M. The observation that 1 M ATP leads to maximal PKD1-Ser 916 phosphorylation but only a very low level of 32 P incorporation into PKD1 provides

PKD1-Ser 744 /Ser 748 and Ser 916 Phosphorylation Mechanisms
unambiguous evidence that PKD1-Ser 916 phosphorylation is via a mechanism that is distinct from the mechanism that controls autophosphorylation at other sites in the enzyme.
The ATP requirements for PKD1 phosphorylation of CREB and cTnI were examined in parallel, to determine whether substrate phosphorylation correlates with overall PKD1 autophosphorylation (detected as 32 P incorporation into the protein) or PKD1 autophosphorylation at Ser 916 . The immunoblotting studies in Fig. 3A show that 1 M ATP does not support PKD1dependent CREB-Ser 133 or cTnI-Ser 23 /Ser 24 phosphorylation. PKD1 induces a dose-dependent increase in CREB phosphoryl-ation as the ATP concentration increases from 6 to 66 M; cTnI phosphorylation is detected at 20 M and is maximal at 300 M. Fig. 3B shows that overall CREB phosphorylation (detected as 32 P incorporation into CREB) also increases in a dose-dependent manner as the ATP concentration increases from 6 to 200 M. The observation that PKD1 substrate phosphorylation correlates with overall PKD1 autophosphorylation (measured as 32 P incorporation into PKD1), but not with PKD1-Ser 916 autophosphorylation, indicates that the Ser 916 -phosphorylated form of PKD1 is not necessarily active toward target substrates.
A PKD1 autophosphorylation reaction that requires only limiting levels of ATP is predicted to be relatively refractory to inhibition by Gö6976. This prediction was tested directly by comparing Gö6976 inhibition of PKD1 autophosphorylation at Ser 916 (detected by immunoblot analysis) with Gö6976 inhibition of overall PKD1 autophosphorylation (detected as 32 P incorporation into PKD1 by PhosphorImager). Fig. 4A (bottom) shows that 32 P incorporation into PKD1 is quite low at 1 M ATP; 32 P incorporation into PKD1 increases progressively (in parallel with an increase in PKD-Ser 744 /Ser 748 phosphorylation; Fig. 4A, top) as the ATP concentration is increased to 66 M. At 66 M ATP, 32 P incorporation into PKD1 and PKD1-Ser 744 /Ser 748 autophosphorylation are inhibited in a dose-dependent manner by Gö6976; a high level of inhibition is evident at 1-5 M Gö6976. In contrast, PKD1-Ser 916 autophosphorylation is similar at 1, 6, and 66 M ATP, and it is relatively resistant to inhibition by Gö6976. 1-5 M Gö6976 does not block PKD1-Ser 916 autophosphorylation at 66 M ATP. PKD-Ser 916 autophosphorylation at 6 M ATP is blocked by Gö6976 but only at a high inhibitor concentration. An effect of lower Gö6976 concentrations to inhibit PKD1-Ser 916 autophosphorylation is detected only when the ATP concentration is reduced to 1 M.
We next examined whether PKD1-Ser 916 autophosphorylation remains relatively refractory to inhibition by Gö6976 when kinase assays are performed with a heterologous substrate such as cTnI. Fig. 4B shows that PKD1-Ser 744 /Ser 748 autophosphorylation and PKD1 phosphorylation of cTnI are detected at 66 M ATP and at a much lower level at 6 M ATP. These phosphorylation reactions (that are impaired when the ATP concentration is reduced to 6 M) are effectively inhibited by 1-5 M Gö6976. In contrast, PKD1-Ser 916 autophosphorylation is blocked only by a high Gö6976 concentration when assays are performed with 6 M ATP. Similar results were obtained in assays with CREB as substrate (data not shown). Collectively, these studies provide novel evidence that the ATP requirements for PKD1 autophosphorylation at the activation loop (Ser 744 and Ser 748 ) and PKD1 trans-phosphorylation of heterologous substrates such as cTnI or CREB are considerably higher than the ATP requirement for PKD1-Ser 916 autophosphorylation.
PKD1 autophosphorylation can be mediated either by a cis intramolecular reaction or a intermolecular phosphorylation in trans. GFP-tagged WT-PKD1 and HA-tagged KD-PKD1 (which have different mobilities and can be resolved by SDS-PAGE) were heterologously overexpressed in COS-7 cells, immunoprecipitated, and subjected to in vitro kinase assays with varying amounts of ATP to determine whether the ATP

PKD1-Ser 744 /Ser 748 and Ser 916 Phosphorylation Mechanisms
requirements for the cis WT-PKD1-Ser 916 autophosphorylation reaction and WT-PKD1 phosphorylation of KD-PKD1 (an obligate trans-phosphorylation) can be distinguished. In vitro kinase assays were performed with buffers containing [ 32 P]ATP to examine whether the KD-PKD1 enzyme preparation might contain contaminants with kinase activity. Fig. 5 shows that assays with KD-PKD1 do not lead to any 32 P incorporation into KD-PKD1 or possible co-precipitating proteins; KD-PKD-Ser 916 phosphorylation also is not detected by immunoblot analysis under these conditions. In contrast, kinase assays with the WT-PKD1 preparation results in the appearance of a single prominent radiolabeled band that co-migrates with the WT-PKD1 enzyme; no contaminating enzymes or substrates are detected in the WT-PKD1 preparation. WT-PKD1-Ser 916 autophosphorylation is detected in assays with 1 M ATP. WT-PKD1 also phosphorylates KD-PKD1 at Ser 916 in trans, but this is detected only when the ATP concentration is increased to 6 -66 M. The failure to detect an intermolecular KD-PKD1 phosphorylation by WT-PKD1 at 1 M ATP suggests that the very efficient WT-PKD1-Ser 916 autophosphorylation at 1 M ATP is mediated by a cis intramolecular autocatalytic reaction.
Autophosphorylation of PKD1-S744E/S748E and PKD1-S744A/ S748A Enzymes-The in vitro controls of PKD1 activity identified in our studies conflict with two of the major prevailing assumptions regarding the mechanisms that contribute to PKD1 activation. First, conventional models view PKD1 phosphorylation as a hierarchical process that is initiated by a phosphorylation reaction at the activation loop that primes the enzyme for a subsequent autophosphorylation at Ser 916 . Our results showing that PKD1 executes a Ser 916 autophosphorylation reaction at 1 M ATP, under conditions that do not support (and do not require) activation loop autophosphorylation, are at odds with this formulation. Second, although there is evidence that activation loop phosphorylation is required for PMA-dependent PKD1 activation in a cellular context, previous studies with a PKD1-S744A/S748A mutant were interpreted as evidence that activation loop phosphorylation is not required for in vitro PKD1 activity (21). This conclusion is surprising, based upon a substantial literature that implicates activation loop phosphorylation as a modification that is essential for the catalytic competence of other Ser/Thr kinases. Our observation that PKD1 activity toward target substrates such as CREB and cTnI increases in parallel with an increase in PKD1 activation loop autophosphorylation (Fig. 3) provided the rationale to revisit studies that consider a possible role for activation loop phosphorylation in the control of PKD1 catalytic activity.
We performed in vitro kinase assays with WT-PKD1, PKD1-S744E/S748E, and PKD1-S744A/S748A to directly examine the role of activation loop phosphorylation in the mechanism leading to PKD1-Ser 916 autophosphorylation and PKD1 phosphorylation of target substrates in trans. Fig. 6A shows that WT-PKD1, PKD1-S744E/S748E, and PKD1-S744A/S748A were heterologously overexpressed in HEK293 cells at similar levels. PKD1-S744E/S748E is recovered from resting HEK293 cells as a constitutively Ser 916 -phosphorylated enzyme that undergoes little-to-no further increase in Ser 916 phosphorylation when subjected to in vitro kinase assays (Fig. 6B). However, PKD1-S744E/S748E executes a prominent in vitro autophosphorylation reaction that is detected as an electrophoretic mobility shift in SDS-PAGE (Fig. 6B) and an increase in 32 P incorporation by PhosphorImager (Fig. 6D). Because PKD1-S744E/ S748E harbors activation loop phosphomimetic substitutions and PKD1-S744E/S748E is constitutively phosphorylated at Ser 916 , the electrophoretic mobility shift and increased 32 P  incorporation must denote PKD1 autophosphorylation elsewhere in the protein. We used a mutagenesis approach to map the structural requirements for the band shift. Fig. 6C shows that a PH domain deletion mutant (PKD1-⌬PH) undergoes a prominent band shift as a result of the in vitro kinase assay, whereas a PKD1 N-terminal truncation mutant lacking the C1 domain (PKD1-⌬C1) does not. These results indicate that the band shift is because of an autophosphorylation reaction that maps to (or is dependent upon) the N terminus of the enzyme. Of note, PKD1 autophosphorylation sites have been mapped to Ser 205 /Ser 208 and Ser 219 /Ser 223 in the C1A/C1B interdomain (22). Although these sites are deleted in the PKD1-⌬C1 mutant, additional studies showed that S205A/S208A-and S219A/ S223A-substituted forms of PKD1 undergo pronounced mobility shifts during in vitro kinase assays (data not shown). These results suggest that PKD1 autophosphorylates at yet additional sites; efforts to map the autophosphorylation sites(s) underlying the mobility shift are ongoing. Fig. 6, B and E, shows that PKD1-S744E/S748E is a robust CREB and cTnI kinase. Maximal CREB-Ser 133 and cTnI-Ser 23 / Ser 24 phosphorylation by immunoblot analysis (Fig. 6B) or 32 P incorporation into CREB or cTnI (by PhosphorImager analysis, Fig. 6E) is considerably higher in assays with PKD1-S744E/S748E than in assays with WT-PKD1. Of note, a S744E/S748E substitution leads to an increase in PKD1 autophosphorylation measured as 32 P incorporation into PKD1 and enhanced PKD1 phosphorylation of heterologous substrates in trans, but it does not result in increased PKD1-Ser 916 autophosphorylation. Fig. 6B shows that the PKD1-S744E/S748E enzyme promotes CREB-Ser 133 phosphorylation at 1-6 M ATP, under conditions that do not lead to a detectable increase in CREB-Ser 133 phosphorylation by WT-PKD1. Although these results could suggest that activation loop Ser 3 Glu substitutions lower the ATP requirement for CREB phosphorylation by PKD1, a Ser 3 Glu substitution that increases overall CREB-Ser 133 phosphorylation might lead to an apparent left shift of the curve by bringing a subthreshold CREB-Ser 133 phosphorylation reaction at 1-6 M ATP into the detectable range. To avoid a result that might be an artifact of the detection method, CREB phosphorylation also was tracked by Phosphor-Imager analysis. Fig. 6, E and F, shows that activation loop Ser 3 Glu substitutions increase overall 32 P incorporation into CREB and also decrease the ATP requirement for CREB phosphorylation by PKD1; this left shift in the dose-response curve for ATP is most evident in Fig. 6F, where the data are normalized for differences in maximal WT-PKD1 and PKD1-S744E/S748E activity.
A PKD1-S744E/S748E mutant with a reduced ATP requirement should be relatively refractory to inhibition by Gö6976. This prediction was tested by comparing the dose-response curves for Gö6976 inhibition of WT-PKD1 and PKD1-S744E/ S748E. Fig. 7A shows that WT-PKD1 executes autophosphorylation reactions at the activation loop (Ser 748 ) and at Ser 916 and that Gö6976 selectively prevents PKD1 autophosphorylation at Ser 748 but not Ser 916 . WT-PKD1 also phosphorylates CREB and cTnI; this is detected as an increase in anti-CREB-Ser(P) 133 and anti-cTnI-Ser(P) 23 /Ser(P) 24 immunoreactivity by Western blot analysis (Fig. 7A) and an increase in 32 P incorporation by PhosphorImager analysis (Fig. 7B). By either measure, WT-PKD1 phosphorylation of CREB or cTnI is abrogated by 5 M Gö6976. PKD1-S744E/S748E is recovered as a constitutively Ser 916 -phosphorylated enzyme that is not recognized by the anti-PKD1-Ser(P) 748 antibody. PKD1-S744E/S748E promotes a high level of CREB-Ser 133 and cTnI-Ser 23 /Ser 24 phos-

PKD1-Ser 744 /Ser 748 and Ser 916 Phosphorylation Mechanisms
phorylation (Fig. 7A). Gö6976 inhibits PKD1-S744E/S748E phosphorylation of CREB and cTnI, without blocking PKD1-S744E/S748E-Ser 916 autophosphorylation. However, the effect of Gö6976 to inhibit PKD1-S744E/S748E activity requires high inhibitor concentrations, ϳ4-fold higher than the Gö6976 concentration sufficient to inhibit WT-PKD1 (Fig. 7B). These results expose a potential limitation of PKD1 inhibitors that act by competing for ATP binding; the efficacy of these compounds will be influenced by the activation state of (and post-translational modifications on) the enzyme.
Studies of the PKD1-S744A/S748A mutant also were revealing. Fig. 6B shows that the PKD1-S744A/S748A enzyme autophosphorylates at Ser 916 , similar to WT-PKD1. However, PKD1-S744A/S748SA incorporates relatively little 32 P; it does not band shift, and it is a very ineffective CREB-Ser 133 or cTnI-Ser 23 /Ser 24 kinase when subjected to in vitro kinase assays with PMA (Fig. 6B, D, and E). These results provide direct evidence that PKD1 does not require a phosphorylation or negative charge in the activation loop to execute an autophosphorylation reaction at Ser 916 . The results also suggest that a phosphorylation or negative charge in the activation loop is required for PKD1 activity toward target substrates. Although this conclusion resonates with prevailing concepts regarding the mechanisms regulating the activity of various other Ser/Thr kinases, it is at odds with results published by the Rozengurt laboratory showing that a S744A/S748A substitution does not interfere with in vitro PKD1 activity (21). Because conclusions from the Rozengurt laboratory were based upon in vitro kinase assays performed with a different lipid cofactor (PDBu, rather than PMA) and a peptide (syntide-2) rather than a protein substrate, we performed additional kinase assays to reconcile discrepancies between this and the previous study. Fig. 8 shows that WT-PKD1 executes similar robust autophosphorylation reactions, detected as either Ser 916 phosphorylation (Fig. 8A) or 32 P incorporation into the enzyme (Fig. 8B), when in vitro kinase assays are performed with either PDBu, PMA, or dextran sulfate (a compound used by the Rozengurt laboratory as a potent activator of PKD1 (21)). WT-PKD1 activity toward syntide-2 also is increased by lipid cofactors and dextran sulfate (Fig.  8D). In contrast, the magnitude of the lipid cofactor-dependent increase in WT-PKD1-dependent CREB phosphorylation is quite modest when compared with the massive dextran sulfate-induced increase in CREB kinase activity. These results suggest that the controls of WT-PKD1 activity toward peptide and protein substrates might differ. Fig. 8A shows that the PKD1-S744A/S748A mutant autophosphorylates at Ser 916 when kinase assays are performed with lipid cofactors or dextran sulfate. Although basal 32 P incorporation into PKD1-S744A/S748A is reduced relative to basal 32 P incorporation into WT-PKD1, lipid cofactor-dependent increases in 32 P incorporation into WT-PKD1, and PKD1-S744A/S748A enzymes are quite similar (Fig. 8B). However, the absolute level of lipid-dependent syntide-2 kinase activity for the PKD1-S744A/S748A mutant was quite low compared with WT-PKD1 (Fig. 8D). Moreover, the PMA-activated PKD1-S744A/S748A enzyme is autophosphorylated at Ser 916 , but it does not act as a CREB kinase. However, the PKD1-S744A/ S748A enzyme exhibits a band shift, and PKD1-S744A/S748A becomes an effective CREB and syntide-2 kinase (with activity comparable with WT-PKD1), when assays are performed in the presence of dextran sulfate. These results indicate that activation loop Ser 3 Ala substitutions interfere with PKD1 activation by lipids, but they do not lead to structural changes that inherently disable the enzyme.
PKD1-Ser 748 Autophosphorylation Is a Hierarchical Process That Requires a Negative Charge at Ser 916 -Having demonstrated that PKD1 does not require a phosphorylation reaction (or negative charge) at the activation loop to autophosphorylate at Ser 916 , the final set of studies used a mutagenesis strategy to examine whether Ser 916 autophosphorylation exerts a reciprocal effect to regulate PKD1 activation loop autophosphorylation and/or PKD1 activity. Fig. 9A shows that the PKD1-S916A enzyme undergoes an autophosphorylation reaction that is detected as an electrophoretic mobility shift and an increase in immunoreactivity for the Cell Signaling Technology anti-PKD1-Ser(P) 744 /Ser(P) 748 PSSA, similar to WT-PKD1. However, 32 P incorporation into PKD1-S916A (at maximal ATP) is reduced by 37.8 Ϯ 6%, compared with 32 P incorporation into WT-PKD1 (n ϭ 3, Fig. 9B). Although this presumably is attributable to the loss of Ser 916 as a phosphoacceptor site, our studies expose an additional effect of the S916A substitution to induce a secondary defect in PKD1 autophosphorylation at Ser 748 . Additional studies show that WT-PKD1 and PKD1-S916A enzymes elicit similar increases in CREB and cTnI phosphorylation, measured as increased CREB-Ser 133 and cTnI-Ser 23 /Ser 24 phosphorylation by immunoblot analysis (Fig. 9A) or 32 P incorporation into CREB and cTnI by PhosphorImager analysis (Fig.  9B). These results indicate that a PKD1-Ser 748 /Ser 916 autophosphorylation defect does not impair in vitro PKD1 activity toward protein substrates.
WT-PKD1 and PKD1-S916A were heterologously overexpressed in HEK293 cells to determine whether Ser 916 phosphorylation also regulates PKD1-Ser 748 phosphorylation and PKD1 activity in vivo in a cellular context. Fig. 10 shows that PMA treatment leads to a similar mobility shift and increase in Ser 744 /Ser 748 phosphorylation for WT-PKD1 and PKD-S916A enzymes. In contrast, PMA treatment increases Ser 748 phosphorylation on the WT-PKD1 enzyme, but PMAdependent Ser 748 phosphorylation is markedly reduced in the PKD1-S916A enzyme. The S916A substitution also influenced PKD1 activation by thrombin, an agonist for the G protein-coupled protease-activated receptor. Thrombin triggers similar increases in WT-PKD1 and PKD1-S916A phosphorylation at Ser 744 /Ser 748 ; thrombin-dependent Ser 744 /Ser 748 is maximal at 5 min and wanes when the stimulation interval is prolonged to 30 -60 min. In contrast, thrombin induces biphasic increases in WT-PKD1 phosphorylation at Ser 748 and Ser 916 . Phosphoryla-  tion at these sites increases at 5 min and wanes as the stimulation interval is increased to 30 min. However, a secondary increase in PKD1-Ser 748 /Ser 916 phosphorylation is detected when treatment with thrombin is prolonged to 60 min. The effect of thrombin to promote PKD1-Ser 748 phosphorylation (at all time points) is abrogated by the S916A substitution.
The final series of studies examined whether the S916A substitution influences in vivo PKD1 activity toward physiologically relevant substrates such as CREB and HSP27. Fig 10 shows that PMA and thrombin increase CREB-Ser 133 and HSP27-Ser 82 phosphorylation in control ␤-galactosidase-overexpressing cultures. PMA induces a sustained increase in HSP27-Ser 82 phosphorylation that persists for at least 60 min, whereas thrombin-dependent HSP27-Ser 82 phosphorylation is maximal at 5 min and wanes when treatment with thrombin is prolonged to 60 min. WT-PKD1 overexpression increases the magnitude, without changing the kinetics, of the thrombin-dependent HSP27-Ser 82 and CREB-Ser 133 phosphorylation responses. In contrast, thrombin-dependent CREB-Ser 133 and HSP27-Ser 82 phosphorylation persists for at least 60 min in PKD1-S916A overexpressing cultures. Thrombin treatment for 60 min leads to a 6.2 Ϯ 0.5-fold higher CREB-Ser 133 phosphorylation and a 3.1 Ϯ 0.03-fold higher HSP27-Ser 82 phosphorylation, in PKD1-S916A overexpressing cultures than in WT-PKD1 overexpressing cultures (n ϭ 3, p Ͻ 0.05). Collectively, these data indicate that a PKD1-S916A substitution leads to a defect in PKD1 autophosphorylation at Ser 748 and that PKD1-Ser 916 and/or Ser 748 autophosphorylation plays a heretofore unrecognized role to terminate the cellular PKD1 signaling response.

DISCUSSION
PKD1 has recently emerged as a signaling enzyme that is activated by many physiologically important stimuli and contributes to growth, survival, and functional responses in many cell types. PKD1 activity is tightly regulated in vivo by phospho-rylation at two highly conserved serine residues in the catalytic domain activation loop. Current dogma holds that PKD1 activation loop phosphorylation is mediated by PKC and is followed by PKD1 autophosphorylation at Ser 916 . Studies reported herein challenge several aspects of this dogma, showing the following: 1) PKD1 executes an autophosphorylation reaction at the activation loop; 2) PKD1-Ser 748 autophosphorylation is a hierarchical process that requires prior phosphorylation at Ser 916 ; and 3) PKD1 autophosphorylation at Ser 916 is mediated by a unique catalytic mechanism that proceeds at exceedingly low ATP concentrations, does not require prior PKD1 phosphorylation at Ser 744 /Ser 748 , and is not necessarily accompanied by increased PKD1 activity toward heterologous substrates. We also show that PKD1-Ser 916 phosphorylation can be mediated by either an intra-or inter-molecular reaction, further undermining the conclusion that PKD1-Ser 916 autophosphorylation provides a valid readout of the phosphorylation state of that particular PKD1 molecule.
Although PKD-Ser 916 autophosphorylation is generally touted as a valid surrogate measurement of PKD activity, discrepancies between PKD1-Ser 916 phosphorylation and measurements of PKD activity are quite common in the literature (16 -18). These inconsistencies are not readily reconciled by established models of PKD1 activation and have generally been ignored. This study presents a series of observations that challenge the prevailing assumption that PKD-Ser 916 autophosphorylation provides a valid surrogate measure of PKD activity under all experimental conditions. First, we show that KD-PKD1 (which by definition cannot undergo Ser 916 autophosphorylation) is recovered from cardiomyocytes as a Ser 916phosphorylated enzyme. Of note, KD-PKD1-Ser 916 transphosphorylation by a GF109203X-sensitive signaling pathway also was previously identified in B lymphocytes (23) and may be a common feature of cells with relatively high endogenous PKC/PKD activity. Second, we show that Gö6976 effectively blocks PKD-dependent phosphorylation of cTnI or CREB, but this is not associated with a coordinate inhibition of PKD1-Ser 916 autophosphorylation. Although this observation was initially surprising, it is interesting to note that Gö6976 is widely used to implicate PKD in various signaling responses, but experimental evidence showing that Gö6976 inhibits agonistdependent PKD-Ser 916 autophosphorylation is conspicuously absent from the literature. Third, we show that WT-PKD1 autophosphorylates at Ser 916 , but WT-PKD1 does not phosphorylate heterologous substrates when in vitro kinase assays are performed with very low ATP concentrations. Fourth, we show that the PKD1-S744A/S748A mutant autophosphorylates at Ser 916 , but it does not phosphorylate heterologous substrates such as CREB or cTnI. Collectively, these results provide clear evidence that PKD1 autophosphorylation at Ser 916 is a superbly efficient reaction that proceeds under conditions that do not support PKD autophosphorylation at other sites or PKD1 phosphorylation of heterologous substrates; these results argue that PKD1-Ser 916 autophosphorylation does not necessarily provide a valid surrogate measure of PKD1 activation in all cellular contexts.
PKD is rapidly phosphorylated at Ser 744 /Ser 748 in response to various physiologic stimuli in many cell types. This rapid FIGURE 10. The PKD1-S916A mutant exhibits a Ser 748 phosphorylation defect and a prolonged signaling response in HEK293 cells. WT-PKD1, PKD1-S916A, and ␤-galactosidase were heterologously overexpressed in HEK293 cells that were then treated with vehicle, 300 nM PMA, or 1 unit/ml thrombin for the indicated intervals. Cell extracts were subjected to immunoblot analysis for PKD1, CREB, and HSP27 phosphorylation and protein expression. Results are representative of data obtained in three separate experiments.

PKD1-Ser 744 /Ser 748 and Ser 916 Phosphorylation Mechanisms
increase in PKD-Ser 744 /Ser 748 phosphorylation has been ascribed to a nPKC-dependent mechanism based upon a large number of studies showing that PKD1-Ser 744 /Ser 748 phosphorylation is effectively blocked by PKC inhibitors (that do not directly inhibit PKD activity). However, it is important to note that there is only very limited direct in vitro experimental evidence that nPKC isoforms phosphorylate PKD1 at Ser 744 and/or Ser 748 (24). A model that implicates PKCs as obligatory PKD1 activation loop kinases also is difficult to reconcile with studies from the Rozengurt laboratory showing that the PKD1-⌬PH mutant is recovered from GF109203X-treated COS-7 cells as a constitutively active Ser 744 /Ser 748 -phosphorylated enzyme (25,26). These results suggest that PKD1-⌬PH-Ser 744 / Ser 748 phosphorylation is mediated by a PKC-independent mechanism. In fact, the Rozengurt laboratory has recently revisited this issue and demonstrated that PKD1-⌬PH activation loop phosphorylation can be mediated by an autocatalytic reaction (5). This study provides direct evidence that the PKD1 activation loop is phosphorylated via an autocatalytic mechanism that may contribute to the physiological control of PKD1 activity when PKC isoforms are down-regulated.
Current concepts regarding the mechanisms that regulate PKD1 activation loop phosphorylation are based almost exclusively on studies that used the anti-PKD1-Ser(P) 744 /Ser(P) 748 PSSA. The notion that this reagent tracks phosphorylation primarily at Ser 744 , and that the controls and consequences of PKD1 phosphorylation Ser 748 may differ, has not been considered. This study exploits the properties of a recently developed PSSA that specifically recognizes PKD1 phosphorylation at Ser 748 to expose striking differences in the mechanisms and consequences of PKD1-Ser 744 and -Ser 748 phosphorylation. First, cell-based studies show that heterologously overexpressed KD-PKD1 displays robust phosphorylation in trans at Ser 744 , but catalytically inactive PKD1 displays only a low level of Ser 748 phosphorylation. These results suggest that in vivo PKD1-Ser 748 phosphorylation is primarily via a cis autophosphorylation reaction that is defective in the catalytically inactive enzyme. Second, this study presents novel evidence that Ser 748 autophosphorylation requires a prior priming autophosphorylation at Ser 916 ; the S916A substitution leads to a PKD1-Ser 748 phosphorylation defect, without any changes in Ser 744 phosphorylation in vitro and in vivo in agonist-activated HEK293 cells. These results provide strong evidence that Ser 748 phosphorylation is mediated by an autocatalytic reaction (rather than a trans-phosphorylation by PKC). These results suggest that the PKD1 C terminus, and particularly the Ser 916 autophosphorylation site, plays a heretofore unrecognized role to structure the kinase core for some aspect of its catalytic function. Although this mode of regulation is novel for PKD1, it is well described for cAMP-dependent protein kinase and PKC, where phosphorylation at the extreme C terminus tethers the C terminus away from the active site and stabilizes the enzyme in a conformation that favors catalysis (27,28). However, Ser 916 / Ser 748 autophosphorylation defects do not lead to gross abnormalities in PKD1 autophosphorylation at other sites (such as Ser 744 or the sites elsewhere in the protein that underlie the electrophoretic mobility shift) or PKD1 phosphorylation of heterologous substrates such as CREB or cTnI. Rather, cell-based studies identify a biphasic thrombin-dependent Ser 916 / Ser 748 phosphorylation response and a more prolonged PKD1 signaling response when Ser 916 /Ser 748 autophosphorylation is prevented (by the S916A substitution). These results suggest autophosphorylation reactions at Ser 916 and/or Ser 748 function to limit the duration of the PKD1 signaling response; these results emphasize that studies that use Ser 916 autophosphorylation as a marker of PKD1 activation may be misleading.
Whereas the distinct controls and consequences for activation loop Ser 744 and Ser 748 phosphorylation identified in this study have not previously been noted for mammalian PKD family enzymes, they resemble features recently described for the Caenorhabditis elegans PKD enzymes, DKF-1 and DKF-2 (29 -31). The DKF-1 activation loop sequence contains a single phospho-acceptor site at QFRKT 588 , corresponding to Ser 748 in PKD1. Thr 588 phosphorylation is via a PKC-independent mechanism and plays a dual role to mediate the PMA-dependent increase in DKF-1 activity and to tag DKF-1 for ubiquitinylation and proteasomal degradation (29,30). The 925 SFRRS 929 activation loop sequence in the other C. elegans PKD enzyme DKF-2 is identical to the 744 SFRRS 748 sequence in PKD1 (31). Phosphorylation reactions at Ser 925 and Ser 929 in DKF-2 have been attributed to a PKC-dependent mechanism, with Ser 925 phosphorylation implicated in the PMA-dependent mechanism that increases DKF-2 activity and Ser 929 phosphorylation implicated as a modification that terminates the DKF-2 signaling response. The results of this study, showing that phosphorylation reactions at Ser 744 and Ser 748 play functionally distinct roles to regulate either the magnitude or the kinetics of the PKD1 signaling response, suggest that these features have been evolutionally conserved in the mammalian PKD1 enzyme.
This study implicates Ser 744 phosphorylation as a modification that is required for PKD1 activity toward heterologous substrates such as CREB, cTnI, and syntide-2. Although these results resonate with considerable literature that implicates activation loop phosphorylation as a modification that controls the catalytic function of other Ser/Thr kinases, the results are at odds with studies from the Rozengurt laboratory that have been interpreted as evidence that the in vitro kinase activities of PKD1-S744A/S748A and WT-PKD1 are indistinguishable. Of note, the discrepancies between this and the previous study are readily attributed to differences in assay conditions and methods of data analysis. Studies from the Rozengurt laboratory focused exclusively on PKD1 phosphorylation of the peptide substrate syntide-2; results for lipid cofactor-dependent enzyme activity were normalized to correct for any differences in basal PKD1 versus PKD1-S744A/S748A activity. Studies reported herein show that lipid cofactor-dependent increments in PKD1-S744A/S748A and WT-PKD1 syntide-2 kinase activities appear comparable when the data are expressed in this manner. However, the normalized data obscure the marked reduction in the absolute level of lipid-dependent syntide-2 kinase activity that results from a S744A/S748A substitution. Moreover, studies reported herein show that a S744A/S748A substitution abrogates lipid-dependent activity toward protein substrates such as CREB and cTnI, exposing an important potential fallacy of studies that rely exclusively on kinase assays

PKD1-Ser 744 /Ser 748 and Ser 916 Phosphorylation Mechanisms
with model peptides to interrogate mechanisms that control PKD1 phosphorylation of physiologically relevant substrates.
Finally, this study shows that PKD1 executes a series of phosphorylation reactions with distinct ATP requirements. PKD1 undergoes an intramolecular autophosphorylation at Ser 916 at a very low ATP concentration; PKD1 autophosphorylation at other sites and PKD1 phosphorylation of target substrates are detected only at considerably higher ATP concentrations. Although intracellular ATP levels are sufficiently high to support all PKD1 phosphorylation reactions under physiologic conditions, these differences in ATP requirements could become relevant in ischemia, where a fall in ATP might lead to a selective defect in the phosphorylation of target substrates such as cTnI and CREB with little-to-no associated defect in PKD-Ser 916 autophosphorylation (a modification that is required for PKD1 interactions with PDZ domain-containing binding partners and PKD1 targeting within the cell). Our studies showing that post-translational modifications such as activation loop phosphorylation lower the ATP requirement for PKD1-dependent phosphorylation of heterologous substrates also may have clinical implications; our results raise the concern that the "therapeutic window" for ATP-competitive inhibitor compounds may vary according to the activation status of the PKD1 enzyme. These results emphasize an inherent limitation of kinase inhibitors that compete with ATP at the binding pocket and the importance of developing novel compounds that act through different mechanisms.